QBM Exam 2 Chapters 7-12

Native Gel Electrophoresis

"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.

29. What is the advantage and disadvantage to using magnetic beads to purify proteins?

(inexpensive, fast, disadvantage is that sample isn't very pure) Magnetic beads can be used directly in microcentrifuge tubes and it is a very fast technique. Researchers would also not need to invest thousands of dollars in a chromatography system. The disadvantage is that there is no associated elution gradient, fractionation, or chromatogram which means the sample will not be as pure as those purified via chromatography.

Indirect ELISA

***indirect ELISA has a setup identical to a western blot in that the antigen bound is to the membrane and a primary antibody is added to the well, followed by a second one***

Analytical Applications of SEC • SEC columns are used analytically for studying

- Protein purity - Protein folding / conformation - Protein-protein interactions - Quaternary structure - Mw with a calibration (standard) curve

4. You are studying p53 (which forms a TETRAMER) and its binding partner ATM (MONOMER) on gels. If the two proteins interact, what would you see on a native gel? On SDS PAGE?

1 band on a native gel of the entire complex, and 2 bands on SDS-PAGE - one for p53, linearized, one for ATM

Two-Dimensional Electrophoresis Applications

2D-electrophoresis of total cell proteins from various organisms can resolve hundreds of different proteins 2D Applications • Physiological conditions - Cancer-specific proteins - Heart-disease specific proteins - Heat shock proteins • Mutations/variants - Mutant proteins - excise spot, N-terminal sequencing or mass spectrometry for identification, cloning, etc. - Splice variants • Proteomics - Gene-protein index

Buffer used in native gel electrophoresis is usually around pH ____.

9! so most proteins will carry a negative charge and migrate toward the positively charge electrode. This is not always the case, and proteins with very high pI's will migrate in the reverse direction towards the negative electrode.

3. How does a western compare to a far western and southwestern blot?

A far western utilizes a radiolabeled protein probe, not an antibody A Southwestern utilizes DNA as a probe instead of an antibody.

27. Name three reasons why metal-chelate chromatography is advantageous.

A his tag is easily inserted onto the amino/carboxyl terminus of a protein via PCR, and does not disrupt protein function so it doesn't need to be cleaved. It is one of the best forms of purificarion, often sufficiently purifying proteins in a single step. Lastly, Anti-his bodied can also be used to locate the protein on a western blot.

3. What is the major advantage to size exclusion chromatography, and why is it often used as a final step in protein purification?

A major advantage to size exclusion chromatography, making it a last step in protein purification is because.... it is a gentle purification technique, it can remove protein aggregates, and it acts as a buffer exchange.

1. What are the advantages of a western blot over SDSPAGE when identifying a protein of interest on a gel?

A western blot adds a second level of identification, the molecular weight was known from the SDS-PAGE gel, but the band on the gel could be bacterial or other contaminants. A western blot uses an antibody specific for the protein of interest, so any visible bands on western at the corret molecular weight essentially GUARANTEE that the protein you are seeing is the target protein. Western blots are also more sensitive can can detect very low concentrations of proteins that may not be visible on an SDS-PAGE GEL.

7. What is the purpose of each of the following in an SDS-PAGE gel?

Acrylamide- forms long polymers BIS- forms crosslinks between acrylamide chains APS- provides the free radicals necessary for polymerization TEMED- assists in the transger of free radicals to this BID/Acrylamide for polymerization Stacking Gel- allows proteins to concentrte before reaching the resolving gel Resolving Gel- which actually separates the proteins

14. What is the major advantage of an ammonium sulfate cut over recombinant DNA technology?

Ammonium sulfate can be used to purify proteins directly from a cell extract or organ and new proteins can be discovered in their native state. Recombinant DNA technology can be efficiently used on known genes to express proteins, but it NOT as useful for find NOVEL PROTEINS

15. How does an ammonium sulfate cut differ from using urea or guanidine?

Ammonium sulfate is a structure-forming salt and retains the protein's tertiary structure, whereas UREA and GUANIDINE are chaotropic and linearize the protein to its primary structure. They can then be removed over a column or dialysis in an attempt to refold a denatured protein or inclusion body

10. An ELISA is very sensitive, and may produce positive hits that are not detectable by western blot. Why then must an ELISA be verified with western?

An ELISA may produce false positives because similar antibodies can cross-react with other proteins A western blot provides the molecular weight of the protein being bound by the antibody in the ELISA, and therefore the western will rule out any nonspecific binding.

11. How does a pull-down work?

An antibody bound to a bead binds to a target protein in a lysate. This target protein MAY interact with binding partners, which would therefore be "pulled down" with the target proteion

Immunoprecipitation and Co-Immunoprecipitation

Antibodies can also be used in the purification of a specific protein

Antibodies

Antibody - protein that binds to an epitope • Specificity - ability to bind to specific target with low background • Affinity - how tightly the Ab binds to the target • Polyclonal - Multiple epitopes, lower specificity, higher affinity/signal, more tolerant of different fixation conditions - Produced by injection into animals (faster, cheaper • Monoclonal - Single epitope, high specificity, lower affinity/signal, sensitive to fixation conditions for tissue specimens - Produced from hybridomas • In some circumstances the polyclonal Ab may be more useful than monoclonal Ab for immunohistochemical studies of tissues for morphological examination • Neither can be used inside living cells (150 kDa, 4 chains) • Nanobodies - camel, shark, llama antibodies - only 2 heavy chains, continuous binding site, 12 kDa, can be expressed inside of cells. Shark antibodies can be boiled or put in urea and remain active

antibodies are produced from

B-cells in the blood and lymphatic systems. These B-cells can be isolated and fused with cancer cells to make hybridomas, which produce only a single antibody, called a monoclonal antibody, that targets a single epitope. These antibodies have high specificity because they generally will not cross-react with other proteins, but they will likely have lower affinity and tend to be more sensitive to fixation conditions for true specimens. This is very advantageous in some assays such as ELISA when specificity is essential.

4. Why can SEC be used as a buffer exchange?

Because the proteins will fall through the beads faster than the original buffer They will be transferred to the buffer that is equilibrated on the column.

ANTIBODIES CAN BE DETECTED VIA

CHROMOGENIC REACTION, AUTORADIOGRAPHY, CHEMILUMINESCENCE, OR DIRECT INFRARED FLUORESCENCE Detection is EXTREMELY sensitive, down to the nanogram and picogram rando a Coomassie stained SDSPAGE gel may only detect micrograms of proteins, and is not specific for a single protein of interest like western blottin. Western blotting, on the other hand, can detect picograms of a specific protein out of thousands of proteins on a gel

Oligomeric Studies

Can analyze protein oligomeric state by native gel electrophoresis

30. Why would a researcher use chitin column over a GST or nickel column?

Chin binds intein, the tag, and the protein of interest can be eluted by adding DDT to the column to induce disulfide bond breakage. Very efficient and clean. Chitin in insects removes protein of interest and leaves pure sample. This is an efficient way to remove the tag from the protein of interest

5. Primary antibodies that are conjugated to HRP exist? Why then do researchers use the primary/secondary system?

Conjugating HRP to an antibody takes some work and organic chemistry, it is not feasible for every antibody needed. Therefore, researchers usually conjugate a single secondary antibody to HRP and use this to target all primary antibodies from another species. For example, if a researcher discovers a new protein they can inject it into a goat and obtain primary antibodies. They can then run the protein on a gal, wash i with primary antibody, and wash it with a secondary antibody, from rabbits, conjugated to HRP that targets the Fc of all goat primary antibodies. This also allows one to VISUALIZE multiple proteins and primary antibodies on a gel with a single SECONDARY protein.

A SPECIAL TYPE OF Native Gel Electrophoresis is Pore-Limited Gel Electrophoresis

DOES ALLOW FOR AN ESTIMATION OF MOLECULAR WEIGHT A resolving gel of approx 3-25% acrylamide is poured with no SDS No stacking gel is used • Proteins migrate through the gel until they reach a point where the pore size of the medium is too small and they cannot migrate further • Thereafter, the individual proteins become focused at the point where the pore size becomes limiting • The molecular weight of an unknown protein can be estimated by comparing to protein markers

26. What is the disadvantage of the gene fusion technique?

Disadvantage of the gene technique - the protease cleavage step can be time-consuming and may degreade the protein of interest, it adds extra chromatography steps to the purification to remove the tag which reduces yield, and the protein of interest's solubility may be reduced and it may be more likely to denature or precipitate when the highly-soluble tag is removed.

9. Describe the phage expression system once IPTG is added to the media. Which enzyme transcibes the gene of interest into mRNA?

E.Coli takes up IPTG and it binds to the LAC REPRESSOR, which REMOVES the repressor from the OPERATOR. E.coli RNA polymerase synthesis T7 RNA polymerase which targets the t& promotor on the plasmid and transcribes mRNA from the gene of interest. E.coli ribosomes translates mRNA into protein.

23. What is the purpose of each of the follow in the final protein buffer?

EDTA- chelates metals the proteases use to cleave proteins beta-Mercapoethanol/DDT- prevent oxidation Sodium Azide- inhibit bacterial and fungal growth Glycerol- prevents ice crystal formation which can damage proteins

One of the first techniques developed for protein sequencing is Edman Degredation limited to ~50-60 amino acids of a protein, but is generally only used to sequence the first 10 amino acids, which should provide enough information to identify the protein being sequenced.

Edman degredation, which involves N-terminal sequencing of a very pure protein sample. The bands can be cut directly from a gel or blot, but one must be careful as keratin from skin cells can easily contaminate the sample.

12. Can a protein be purified with both anion and cation exchange chromatography, or only one of the two?

Either can be used, but the pH must be adjusted to get the protein to bind to one or the other.

5. In the Yeast-two-hyrid, the activating and binding domain of GaI4 must be part of the same polypeptide chain for transport, true or false?

FALSE The proteins can be synthesized independently, they only need to both be present and interact with one another for RNA transcription, an thus a color change occurs

24. Why are tags such as the FLAG tag useful?

FLAG tags are small so they shouldn't affect protein function and this tag can be targeted by antibodies in various techniques such as affinity chromatography. This saves researchers time and money as they don't need to generate or purchase antibodies to their specific protein, only to the tag which can be used across multiple samples.

7. What information can be gathered from ITC?

Free energy dG, enthalpy dH, entropy dS, dissociation constant D, and molar ratios of protein-protein interactions

11. Rank the following from best to worst with regards to lysing bacterial cells.

French Press Sonication Chemical Lysis Lysozyme Freeze/Thawing Mortar and Pestle or Blender

Some peaks can overlap which indicate there are multiple proteins in a sample not separated sufficiently; it can be difficult to determine where one peak ends and another begins. What can one do?

Further Analyze fractions on a gel, or SDS-PAGE, which can verify the presence of the wanted protein and its purity relative to contaminants. Also, an enzyme assay may be used if the protein of interest is tagged to an enzyme, like GST for example

1. Why are gas chromatography and thin layer chromatography not used for protein purification?

Gas chromatography is harsh and would denature proteins. Thin layer chromatography uses harsh organics and is also an analytical technique and would thus not provide much sample for purification.

23. Name three reasons why the gene fusion technique is advantageous.

Gene fusion technique is advantageous because -fusion genes can be purified on special columns, such as GST -they can increase solubility -they allow for protein tracking -they can be identified on a western blot with an antibody

All Affinity techniques discusssed involve weak interactions,

HALOTAG(Promega) actually involve a tag on proteins that will form a covalent bond with the column, this column will provide the best purification as it can be washed indefinitely and eluted when one wishes with a protease, Protease has a his-tag and can be removed from the solution by passing it over a Nickel Column

Pregnancy test, Sandwich ELISA

Human chorionic gonadotropin is produced from the developing placenta and found in urine. A pregnancy test has anti-hCG antibodies located at the test strip, and this results in a chromogenic reaction is hCG and two antibodies are present

6. If human proteins are desires, why aren't human cells uses to express proteins?

Human-Mammalian cells are difficult to work with compared to bacteria, and they have special media and incubator requirements. These systems have not been optimized.

What the feck is HIC?

Hydrophobic Interaction Chromatography (HIC) Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions.

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction Chromatography is used ti separate proteins based on their lack of charge. HIC is used when there are available hydrophobic amino acids on a protein's surface. Hydrophobic amino acids are usually at the protein's core away from water, but occaisionally pockets of amino acids are found on the surface where protein may interact with other proteins or the lipid membrane • Aggregation of solute molecules is thermodynamically favorable • Sample placed in high salt buffer (such as ammonium sulfate) which enhances hydrophobic interactions Method is similar to IEX but SALT GRADIENT IS REVERSED, the sample is placed in a buffer that is HIGHLY POLAR(high salt concentrations enhancing hydrophobic interactions) and loaded onto a column. After non-binding proteins have been washed off, bound proteins are eluted by reducing the salt concentration which makes the buffer more non polar. Least hydrophobic proteins elute first, and more hydrophobic proteins are released at lower concentrations of salt.

PTAC -> LacO -> T7 RNA polymerase

IPTG displaces lac respressor from the lac operator, activates t7 polymerase

7. How is the lac repressor used in the Ptac expression system? Which enzyme actually performs transcription of the gene of interest in this system?

IPTG removes the lac repressor from the operator, which allows E.coli RNA polymerase to TRANSCRIBE the gene of interest, and E.coli ribosomes to synthesize the protein of interest.

28. How does imidizole elute the protein of interest from the column?

Imidazole competes with histidine for the nickel-coated beads, which, at some concentration, causes the protein to dissociate and elute from the column.

9. What does each primary and conjugated antibody target in both types of ELISA?

In Indirect ELISA, a primary antibody targets the antigen of interest, and its Fc is tagrte by the conjugates secondary enzyme. In Sandiwch ELISA, both the primary and conjugated secondary enzyme target the antigen of interest

2. mRNA and ribosomes can be used to synthesize proteins in vitro. Why then are prokaryotic systems still the most popular system for protein expression in the lab?

In vitro translation is expensive and yields are low Prokaryotic systems are tried and tested, easy to work with, cost effective, and many protocols have been optimized around these systems over the past few decades.

4. What are inclusion bodies? and why are they a concern?

Inclusion bodies are misfolded protein aggregates that can occur when expressing human proteins in bacterial systems. They are very difficult to resolubize and may render the protein unusable. The only option is to attempt to resolubize them with urea or guanadine

What can be a dead end for purification?

Inclusion bodies can be a dead end for purification and one must redesign and construct, but protein refolding can be attemped VVVV

7. Which type of ELISA is most similar to the western blot?

Indirect ELISA

17. Describe what happens with an amino acid with a pl of 7 as pH decreases from 9 to 5.

Initially, the protein would be negatively-charged. As pH decreases to 7 more hydrogens are present in solution, and the amino terminus will take up hydrogens to go from NH2 to NH3. The protein will be neutral at this point. As the pH decreases to 5 the carboxyl terminus will take up the next hydrogen, turning COO- into COOH. Making an amino acid more acidic makes for lots of hydrogenation.

AFFINITY CHROMATOGRAPHY method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand. Its high specificity makes affinity chromatography one of the best forms of chromatography.

Involves a molecule on a column binding specifically to a molecule in the sample. Usually the first step in any purification, and so specific that it is often the only column necessary to purify a single protein from thousands in a lysate The specificity of these physiologically relevant interactions derives from complementary surfaces on the interacting molecules (hydrogen bonding, van der waals) • Stationary phase - immobilized molecule that binds with high specificity to a single protein • Most molecules wash away, few macromolecules interact with high specificity with the immobilized molecule and are retained on the stationary phase • The selectivity of affinity chromatography is potentially THE HIGHEST OF ANY FORM OF CHROMATOGRAPHY AFFINITY COLUMNS consist of beads that contain immobilized small molecules or proteins that bind with high specificity to a single protein in a lysate. When lysate consisting of thousand of proteins is washed over the column, the majority are not attracted to the stationary phase and are washed away A few proteins will interact with high specificity with the immobilized molecule and are retained on the column. Its high specificity makes affinity chromatography one of the best forms of chromatography.

7. Why is isoelectric focusing not a standard laboratory purification technique?

Isoelectric focusing is not a standard laboratory purification technique because it requires special equipment, ampholytes are expensive, and separating proteins on charge require more tie and work than SDS-PAGE which separates the samples by size and provides more useful information

6. Why is the Yeast-Two-Hybrid assay so important?

It allows the screening of hundreds of potential binding partner with a protein.

9. Why is ChIP such an important development?

It can be used to identify transcription factor binding sites in a specific cell type. There are many applications of the technique (epigenetic modications and transcription factors)

10. Why is 2-dimensional gel electrophoresis so important?

It has been used to identify many new proteins, and can be used to compare protein expression patterns in different cell types, and identify proteins up- or down-regulated under various conditions or disease states.

22. Why is affinity chromatography one of the best techniques for protein purification?

It involves specifically binding the target protein, and unwanted proteins should flow through the column

31. Why is the HaloTag unique? (HALO, think Covalent. Covalent, best purification possible, it's not a weak bond)

It is a covalent linkage to a column which should offer the best purification possible. All other columns discussed involve weak interactions. It also fluoreces.

9. You run a 50kDa and 60kDa protein over an SEC column and see peaks at 50 ml and 40 ml, respectively. If you mixed the two proteins and they interacted with one another, where would you expect to see a peak?

Less than 40 ml since the interacting proteins will be larger than before. They will fall through the column faster than the individual proteins and probably elute ~30ml

2. What are four ways to identify which fractions from a column contain protein?

Look at the chromatogramMix some fractions with Bradford Reagent and look for blue color in Absorbance An enzyme assay (if available) SDS-PAGE Western Blotting

To positively identify proteins, scientists can compare their bands to a ____________, which contains proteins of known molecular weights and concentration.

MARKER Protein markers are generally easily purified proteins, such as BSA or lysozyme, as opposed to DNA markers which are big, "round" numbers. Proteins migrate LOGARITHMICALLY through a gel, which adds a second layer of difficulting when calculating molecular weights of samples.

IN SDS PAGE, The detergent and the heat disrupt the weak interactions that hold proteins together internally, resulting in

MONOMERIC POLYPEPTIDE CHAINS

17. Why must detergents be used when purifying membrane proteins

Membrane proteins have exposed hydrophobic region that fits into the phospholipid bilayer, and they must be purified in detergents to prevent hydrophobic aggregation.

Who studied chlorophyll and developed a liquid-absorption column containing carbonate to separate plant pigments?

Mikhail Tsvet was the first to use the method "chromatography" for "color writin"

Includes Bodies

Misfolded protein aggregates that are not biologically active.

22. Why should protein samples be stores in small aliquots in the freezer as opposed to large volumes?

Multiple rounds of freeze/thawing can damage the proteins due to the ice crystals forming, so smaller aliquots allow one to take out a small fraction of the total protein when necessary without damaging the supply.

7. Which would elute first from an SEC column with G-25 beads - a 50kDa or 60kDa protein?

NEITHER both proteins should be excluded from the beads, so technically both elute from the column going straight through the beads

10. The isoelectric point is where there is no ____________________________ on the protein.

NET CHARGE (isoelectric point means equal amount of positive and negative charge)

Chapter 10 -

Native Gels, Isoelectric Focusing, and 2D Gel Electrophoresis

How does Native Gel differ from SDS PAGE? The migration of proteins in native gels is due to both the charge and the size of the protein, and they separate on their charge-to-mass ratio Native Gel Electrophoresis can't accurately measure a protein's molar weight.

Native gel does not incorporate SDS, Beta-Mercaptoethanol, or stacking gel. Proteins will therefore migrate relative to BOTH SIDE AND CHARGE. While a protein's size does not change, its charge is relative to its pI and the pH of the buffer, and proteins can therefore be negative or positive, or neutral. Proteins in native gels retain their tertiary structure since SDS is not used, so this can even be used as a purification technique, although YIELDS ARE VERY LOW IN NATIVE GEL ELECTROPHORESIS.

3. Why are native gels not always the primary choice for researchers?

Native gels do not tell anything about the molecular weight of the protein, which is very important for identification an analysis. The bands are also thicker and less defined than SDSPAGE gel bands

2. How does SDS-PAGE differ from native Gel Electrophoresis?

Native gels separate based on both size and charge SDSPAGE denatures proteins with SDS, heat, betamercaptoethanol, and protein Mw can be detemined.

SDS gives proteins a _________ charge. SDS also does what to Quaternary Structure of DNA

Negative Also separates multiple subunits of a quaternary structure if connected by noncovalent bonds

Western Blot • Detect and quantify proteins that react with a specific antibody • Western blotting can be used to determine: - If a given antigen is present - Concentration of the antigenic proteins - Physiological state of a protein (phosphorylation)

Often worked in accordance with SDS is technique that allows one to detect and quanitify proteins that react with a specific antibody. Whereas protein identificaton on SDS-PAGE is based on comparison to markers of known molecular weights (and thus requires some estimation Western Blotting is a more definitive identification technique becaue the antibodies usedshould only react with the target protein. Western blotting can be used to determine if a given antigen is present, and the concentration of the antigenic protein. The antibodies used are so specific that they can often distinguish between two different physiological states of a prootein, such as phosphorylation at a specific amino acid. USeful in medical testing (HIV, other viruses, Mad Cow's Disease, Lyme Disease), although many recently replaced by other techniques such as PCR

NaCl Gradient

Once bound to the column, it is washed with buffer to remove any nonspecific aggregation, at this point proteins are eluted with a sodium chloride gradient. Sodium can compete with positively charge proteins bound to a cation exchange column. Chloride can compete with negatively charged proteins bound in a anion exchange column. Proteins will elute based upon the strength of their interaction with their beads - those that are in a buffer with a pH close to their pI will elute first, and proteins in buffers very far from their pI's will elute last. Nacl gradient slowly increases the salt concentration, is used to elute proteins one by one, rather than all at once. Proteins can also be eluted by varying the pH of the buffer, which is difficult to control and may cause proteins to precipitate on the column as they cross to their isoelectric point.

Stacking gel (5% acrylamide) Stacking gel has a lower percentage of acrylamide and is very porous, allowing proteins to migrate very quickly through stacking gel and concentrate into a narrow band prior to reaching the resolcing gel

Polyacrylamide contains two phases, the stacking gel and resolving gel -Resolving gel contains more acrylamide than stacking Stacking el is cast on top of the resolving gel and contains wells created by a comb, and the protein samples will be loaded in to these wells.

Polyacrilamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.

2. What are the pros and cons of using polyclonal over monoclonal antibodies?

Polycolonal antibodies are generally cheaper and strongly bind to the protein of interest, but are more likely to bind nonspecifically to contaminants Monoclonal antibodies are more specific but are more expensive, have a lower binding affinity, and may not be able to withstand certain fixation conditions.

Primary Antibody

Primary Antibody • The membrane is then soaked in a solution containing an antibody to the protein of interest (primary antibody) The choice of primary antibody often comes fown to whatever is available , and often what is least expensive. Many primary antibodies produced in goats, rabbits, or mice, and there are companies that produce thousands of different ypes of antibodies for purchase It is also possible to express antibodies recombinantly and purify them

3. Why are prokaryotic cells, and especially E. coli, used to express most proteins when many other systems exist?

Prokaryotic expression systems have been optimized over the years, they are efficient at taking up foreign DNA, one can go from cDNA to expressed and purified protein in a matter of days, and they are very cost-effective.

2. Proteases are generally the last thing you want near your proteins. Why would you add proteases to a protein sample, and what can you learn from a digestion?

Proteases tend to cleave in accessible regions between domains, so domains con be determined, and conformational changes can be analyzed.

What does native gel electrophoresis allow us to determine?

Protein Conformation Quaternary Structure(Oligomeric State) Analyze its structure/function Study protein-protein interactions Perform Enzyme Assays directly on the gel Study Protein Modifications (phosphorylation, glycosylaiton, activation.

Chapter 7 -

Protein Expression, Purification, Dialysis, and Storage

Chapter 12-

Protein Sequencing, Analysis, and Interactions

Ion Exchange Chromatography IEX better purification, molecular charge

Proteins are separated based on charge, all proteins have an N- and C- terminus and are composed of 20 amino acids, some of which are negatively or positively charged depending on the pH of the buffer. This property is exploited by ion exchange chromatography, IEX, a technique that separates proteins based on their charge. These amino acids can electrostatically bind to a column containing the opposite charge, and then eluted out by an increasing salt concentration or pH. _______ • Proteins are composed of positively and negatively charged amino acids • The proteins in solution have net charges, which can be balanced by counterions • Ions (immobilized on a gel matrix) can electrostatically bind to the charged groups on the proteins • These ions may then be exchanged for ions in an aqueous solution • Because charged molecules bind to ion exchangers reversibly, molecules can be bound or eluted by changing the ionic strength or pH of the eluting solve

Hydrophobic Interaction Chromatography order of elution

Proteins eluted by reducing salt (opposite of ion exchange), the least hydrophobic (most polar) elute first, more hydrophobic proteins elute later

16. You load a protein sample on a cation exchange column at pH 8 and wash away the unbound proteins. Upon adding an NaCl gradient, which proteins elute first? a. Proteins with a pl of 9. b. Proteins with a pl of 7.5. c. Proteins with a pl of 7. d. Proteins with a pl of 11. e. Proteins with a pl of 8.

Proteins with a pI 9 would elute first. Proteins witha pI that equals the pH, pI 8, would be neutral and wouldn't bind to the column Proteins with the pI below the pH, options b an c, would have a nehative charge and would also not bind to the column. Both of these groups of protein would be found in the flowthrough. The preoteins with a pI of 11, option d, are mroe tightly bound and would elute after proteins with a pI of 9, option A.

1. Why is native gel electrophoresis not the first choice when analyzing proteins with regards to molecular weight on a gel?

Proteins with different sizes and charges can migrate to the same position on the gel, so the data is generally unusable for Mw detemination

19. Why is a solubility test important for protein purification?

Putting a small aliquot of protein in various buffers will indicate whether or not the protein is stable or will precipitate in those conditions This way an entire protein stock is not lost

Rf, Relative Mobility It is calculated by dividing the distance the protein migrates by the distance the tracing dye migrates, to determine mW

Relative of a protein can be used for a more accurate molar weight determination. The relative mobility is the ratio of the distance each protein migrates to that of the tracking dye, which has a high charge-to mass ratio and therefore migrates the fastest. It is calculated by dividing the distance the protein migrates by the distance the tracing dye migrates The migration of a protein is negatively proportional to the log of the protein's mass. The top of gel to tracking dye ratio also allows different gels with different protein migration distance be compared.

Coomassie staining

Requires a methanol destain

Isoelectric Focusing IEF

SDS separates by size native gel by charge and size isoelectric focusing by charge A preotein will not move in an electric field when it has a net neutral charge, which occurs when the buffer pH is equal to the protein's pI, here, poteins are separated based by thie pI values a pH gradient is set up within the IEF gel by the inclusion of various buffers and ampholytes, (low molecular weight zwitterions). During "pre-running" phase, each of the ampholytes migrate to a position where the pH equals its pI, establishing a pH gradednt Sample protein is introduced, and electrophoresis is continues until the net current is 0

3. What is the purpose of each of the following when preparing protein for SDS-PAGE?

SDS- removes charge as a factor when proteins migrate on a gel by giving all proteins an equal charge-to-mass ratio by coating them in a negative charge, and helps to break down protein tertiary structure. beta-ME- breaks disulfide bonds Bromophenol Blue- is the tracking die that allows you to see the protein as it is loaded into the gel, and tells you when the run is complete as it runs ahead of the bands Glycerol- makes the sample more dense than the buffer so it stays in the well Loading Control- such as beta-actin, allows you to normalize your samples across lanes in the event that different amounts of sample were originally pipetted into the wells Heat- helps to denature the proteins and breaks weak interactions

SDS-PAGE used for the routine estimation of protein identification, purity, and quantity

SDS-PAGE electrophoresis. SDS-PAGE stands for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and is useful for molecular weight analysis of proteins. SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits. - Track the course of purification of proteins for which there is no convenient enzymatic assay (locate the fractions) - Visualize and track protein, contaminants - Resolving power ~1% mass differences • To simplify the analysis of mixtures of proteins, it is possible to make the separation rely on only the size of the polypeptide chains • Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Chapter 9 - SDS-Page, Protein Visualization, and Band Quantification

SDS-PAGE, Protein Visualization, and Band Quantification

8. Which type of ELISA offers higher specificity? Why?

Sandwich ELISA has higher specificity This tupe of ELISA ONLY the antigen of interest should remain in the well, and the other antigens are washed out. Therefore, it is less likely to produce a false positive. In an indirect ELISA, all antigens bind in the well, and the rate of false positives is higher, BAD. Sandwich ELISA though requires at least two specific antibodies to the target, which ay not always be available.

Silver Staining

Similar to photography, very sensitive

Chapter 8 -

Size Exclusion, Ion Exchange, and Affinity Chromatography.pptx

13. Why are protein bands fixed to the gel? What is the advantage of this, and which technique alleviates this?

So they don't diffuse out of the gel during staining. The disadvantage of fixing proteins in gels is that the gel is no longer usable for downstream applications such as western blotting, so two duplicate gels must often. TCE alleviates this.

14. NaCl is used to elute a sample from a cation exchange column. Which part of NaCl is actually important for the elution?

Sodium Sodium will bind to the negatively-charged beads in Cation exchange Chloride is used in anion exchange to bind to the

Sodium Dodecyl Sulfate (SDS) • Dodecyl sulfate binds strongly to proteins and causes the folded proteins to become denatured due to its negative charge and repulsion into extended "rods" coated with SDS • On average, one dodecyl sulfate molecule will be present for every two amino acids (~1.3g SDS/ g protein)

Sodium Dodecyl Sulfate (SDS) • Each molecule of SDS has a negative charge at the pH used in electrophoresis, causing the net charge of the coated polypeptide chains to be much more negative than normal • Identical charge-to-mass ratios - The charge-to-mass ratios will be nearly identical for different proteins because the SDS coating contributes a large negative charge to the proteins - Therefore the separation of the denatured, SDS-coated proteins will be due almost exclusively to the difference in sizes of the proteins (because of the frictional resistance of the gel) and not their charges

16. Why are solvents generally not used to purify proteins?

Solvents can be used successfully in some cases (resilient proteins and viruses) but in general, SOLVENTS GENERALLY DENATURE target proteins, making them unusable.

Major disadvantage to Native Gel Electrophoresis

THE TECHNIQUE DOES NOT ALLOW FOR ACCURATE DETERMINATION OF MOLAR WEIGHT OF PROTEIN IN QUESTION This is a major downside as MW is one of the best ways to identify the bands on a protein or DNA gel. Another issue is that the bands are wider and less defined than in SDS PAGE and therefore more difficult to resolve and analyze. Proteins in native gels produce long streaks in the gels.

11. How do you calculate a protein's pl?

Take the average all of the individual pI's of all the protein's amino acids

21. How does HIC differ from IEX?

Techniques are essentially opposites from one another IEX separates on charge and HIC on lack of charge, or hydrophobicity. The elution gradients are therefore opposite one another, with IEX increasing salt to elute out proteins, and HIX decreases salt to elute proteins

8. What advantage does the phage expression system offer over the Ptac system?

The PTAC System can be "leaky" and the protein of interest can be expressed at low levels prior to induction The phage expression system has an extra step and PREVENTS BACKGROUND EXPRESSION, and T7 lysozyme will inhibit this even further

Gel data must be recorded for future reference or publications, why? photograph and gel drying

The bands on gels can be fixed and the gels can be dried in cellophane and taped to a lab notebook, or the gel can be imaged via digital camera and printed.

4. Why are some antibodies usable in nearly every antibody-based technique (western, ELISA, IHC, ICC, etc.)?

The best antibodies are to linear conformational epitopes, and would therefore be usable in western blotting, where the protein is denatures, and ELISA and other assays, where the protein is properly folded.

21. You Pipette two proteins, 30 kDa and 40 kDa, that may interact into dialysis tubing. What will be the size of the protein complex if the 2 interact? What size membrane would be an ideal choice to ensure that only the protein complex remain inside the tubing, and the individual proteins pass through the membrane?

The combined protein would be 70 kDa. Therefore a membrnce cutoff of greater than 40kDa but 70 kDa would be best( so probably a ~55kda membrane cutoff).

12. Why won't SDS-PAGE gels work well if you accidentally swap the stacking and resolving gel buffers, which have different pH's?

The current won't run through the gel properly so you will not get correct separation.

Support Medium (Gel)

The electrophoresis of proteins is made possible by a gel, which prevents proteins from migrating too quickly in the electric field, and allows the separation of different proteins to be retained after the current is turned off. Gel characteristics - Strong - Hydrophilic to prevent hydrophobic interactions with the proteins - Uncharged - Stable over a range of temperatures and in various buffers - It should have a carefully controlled and adjustable pore size • Initially paper and starch gels were used to separate proteins, but acrylamide was found to have more desirable properties for protein electrophoresis.

20. You perform cation exchange chromatography and elute the sample with 1 M NaCl. What must be done before this protein can be purified via anion exchange chromatography?

The eluted protein is in a buffer with a pH below is isoelectric point, and there is a high concentration of NaCl in the sample. Both of these factors will prevent the protein from binding an anion exchange column, so dialysis must be used between ion exchange column to increase the pH and remove the salt.

6. Which would elute from an SEC column with G-50 beads first- a 30kDa protein, 40kDa protein, or 10 mM NaCl? Which would elute last?

The first to elute would be is the 40 kDa because it is the largest the last to elute would be the salt

15. Why is an elution gradient of NaCl used in Ion Exchange Chromatography, as opposed to just adding 1 M NaCl to the column (which would be much quicker)?

The gradual increase of salt allows for separation between proteins with low, moderate, and high charges as those with lower charges will elute from the column first. This will create nice peaks on a chromatogram. If instead 1 M NaCl was added to the column directly every protein would elute into the same fraction and there would be no separation.

Milk does what in a Western Blot?

The membrane is BLOCKED by placing it in milk. The blocking step ensures that all of the free protein binding sites on he membrane are NO LONGER AVAILABLE, and the antibody will ONLY BIND TO THE PROTEIN OF INTEREST (sometimes the blocking step is done overnight to improve results)

8. How could isoelectric focusing detect modifications or proteolysis of a protein?

The modifications and proteolysis would cause a change in the protein's isoelectric point. In the case of proteolysis, the protein would actually be losing amino acids which would affect how it migrated on an isoelectric focusing gel

Sandwich ELISA -more specific

The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). A second antibody coupled to an enzyme binds to the antigen at a second location. This type of ELISA is MORE SPECIFIC and will reduce false positives since only the antigen of interest should remain in the well after washing, but it requires multiple antibodies that recognize the target antigen which may not always be available

8. In EMSA you see a shift on a gel. Explain what this shift is and what it indicates.

The shift results from an interaction between DNA and protein, which form a large complex that moves more slowly on the gel in comparison to the control. This indicates that there is a PROTEIN-DNA INTERACTION THAT SHOULD BE FURTHER ANALYZED

4. Explain how the yeast two hybrid system can be used to screen a library?

The yeast two hybrid system can be set up to screen a library for bait and pray. The bait protein protein is bound to the GaI4 binding domain, and a library of target proteins bound to the GaI4 activating domain are screened. If the binding and activating domains are brought into close proxity a color change is observed in the yeast cells. This indicates there was an interaction between the bait and prey, which can be identified and further analyzed.

25. Can tags be removed from proteins? If so, why would you want to do this?

There is often a protease cleavage site between the tag and the protein of interest, so the tag can be removed... It may be removed if the tag interferes with the protein's function in any way, or if the researcher requires a completely pure target protein with no tag

9. Why are gradient gels advantageous?

They allow you to view a wider molecular weight range of protein on the gel than a standard gel.

5. What advantages do yeast, plant, and insect cell expression systems offer? Why aren't they always used?

They can express larger proteins. Proteins are more likely to be properly folded and modified, they are less likely to produce inclusion bodies. Although, they do take more work to perfect than prokaryotic cells, and it ca take weeks to obtain protein as opposed to just days with bacterial systems.

6. You run an SDS-PAGE gel followed by a western blot (shown below.) How many total primary and secondary antibodies did you need to perform this assay?

Three there are only two bands, and thus only two proteins, seen on the western blot this means that two primary antibodies were used, and a single secondary antibody that targets the Fc, primary antibodies could be used

12. Why do researchers use a purification table?

To track each step of a purification and calculate the final yield.

10. Some labs use pGEX vector (with Ptac promoter) in E. coli BL21 cells. What is the flaw in this setup?

Upon induction, IPTG removes the repressor from BL21 genomic DNA, allowing for T7 transcription translation. The PTAC vector does NOT require T7; its repressor is also removed by IPTG, and the gene of interest is transcribed by E. coli polymerases. Therefore using BL21 and T7 polymerase with Ptac promoter is not required, and may not even reduce EXPRESSION EFFICIENCY as the bacterial cell must also devote resources into synthesizing T7 , which is wasteful

Autoradiography

Uses radio-labeled phosphate and X-ray film, the most sensitive technique

TCE

Viewed under UV, molecule binds to tryptophans

Chapter 11-

Western Blot, ELISA, and Immunoprecipitation

How can one reduce false positives in ELISA?

Western blotting first involves SDS-PAGE so the MW of the protein bound by the antibody is known , which adds a second level of identification and greatly reduces false positives.

1. When and why can native gel electrophoresis be used as a purification step?

When the amounts needed are small, and because the protein is not denatured by heating, SDS, or betamercaptoethanol, it should still be properly folded and usable for downstream experiments.

2. Why can native gels detect phosphorylation and other small protein modifications?

While a small change in Mw, a phosphorylation results in a change in the protein's charge, and potentially its conformation. Therefore native gels can be used to detect phosphorylation that may not be resolvable on a SDS-PAGE gel.

1. Can Edman Degradation be used to sequence a 2 kDa peptide?

Yes it dan, a 2 kDa peptide is only ~18 amino acids, so Edman Degredation can be used. (1 kDa is approximately 9 amino acids) Edman Degredation is limited typically to 50-60, but only 10 is usually done because that is enough to identify the protein in question

32. After elution from a Halo Tag column the solution contains the protein of interest and a his-tagged protease. When passing this sample over a nickel column would you collect what binds to the column or the flowthrough?

You would collect the flowthrough, as the protease with its his-tag will bind an the protein of interest will fall through. HALOTAG(Promega) actually involve a tag on proteins that will form a covalent bond with the column, this column will provide the best purification as it can be washed indefinitely and eluted when one wishes with a protease, Protease has a his-tag and can be removed from the solution by passing it over a Nickel Column

A very powerful technique for identifying new protein-protein interaction is called the YEAST TWO HYBRID SYSTEM

a large library of potential partners can be screened. In the event of protein-protein interactions, the system utilizes a REPORTER GENE (LacZ) that is transcriptionally activated and produces a color change in the yeast • Bait + binding domain • cDNA library with special plasmids that contain the activating domain built into the system • Interaction allows RNA polymerase to begin transcription color change

Intein-coated magnetic beads

a protein of interest is tagged to intein, an insect protein that binds to the glucose derivative of chitin, via disulfide bridge and washed over a chitin column DTT, reducing agent, is added to column to cleave disulfide bond and protein of interest elutes from column

The overall purification of a protein can be tracked and analyzed using

a purification table. -records values such as protein concentration, activity, volume, percent yield, and fold purification to determine which purification steps should be kept or removed in future purifications. The purification protocol can then be modified and improved.

Ptac Expression Vector

a simple expression system that makes use of the lac operon This vector has a PTAC promoter that is SILENCED BY THE LAC REPRESSOR PROTEIN The gene of inteest, GST, is downstream of the promotor, but cannot be expressed... This system is turned on with the addition of IPTG, isopropyl-beta-D-thio-galactopyranoside

Chromatogram

a visible record (such as a series of colored bands, or a graph) showing the result of separation of the components of a mixture by chromatography. measured in absorbance, iex also measures salt conductivity

5. You're studying a group of transciption factors that all interact to form a hexamer consisting of a 20 kDa homodimer, a 30 kDa homodimer, and a 40 kDa homodimer.

a.draw this oligomer 20 kDa dimer has two 10 kDa monomers 30 kDa dimer has two 15 kDa monomers 40 kDa dimer has two 20 kDa monomers b. How many bands would you see if you ere to run this sample on a native gel? you would see 1 band on a native gel, the entire hexamer c. How many bands would you see on SDS PAGE? At what molar weights? 3 bands, on SDS page, one at 10 kDa, 15 kDa, and 20 kDa d. if the homodimers were held together by disulfide bridges, and you heated the samples but forgot b-mercaptoethanol, what would you see on SDSPAGE? 3 bands on SDS PAGE, one at 20 kDa, 30 kDa, and at 40 kDa

N,N'-Methylene Bisacrylamide (BIS) -reacts with acrylamide

acrylamide will only polymerize into long chains, so the cross linking agent BIS is necessary to create pores. Acrylamide and BID generally come pre-mixed in the correct ratio from the manufacturer

order in yeast two hybrid (top to bottom)

activating domain binding partner(prey) bait protein binding domain promotor

Calorimetry a technique for analyzing protein-protein interactions

allows for the measurement of minute heat changes in a sample that indicates binding or conformational change

Chromatin Immunoprecipitation ChIP

allows one to identify binding sites of transciption factors and other DNA-binding proteins. The protein's are cross-linked to the DNA in a cell by formaldehyde and the cells are lysed. An antibody-coated magnet bead that targets the protein of interest is added to the sample, and a magnet is used to purify the antibody-protein-DNA complec from the cell extract The protein is digested and the DNA to which it is bound is identified and analyzed using DNA sequencing, PCR, or a microarray

Southwestern Blot search for proteins that bind DNA SDS page, remove SDS, refold with urea, and blotting the gel on nitrocellulose membrane, next digested labeled dna is added, and DNA-binding proteins can be identified.

allows one to search for proteins that bind DNA by using SDS-PAGE to separate the proteins by size, removal of the SDS, refolding of proteins with urea, and blotting the gel onto a nitrocellulose membrane, next digested labeled dna is added, and DNA-binding proteins can be identified.

Native gel Electrophoresis relative size and charge

allows researchers to run proteins on a gel without denaturing them, therefore retaining their native structure and activity.

Ammonium Sulfate though is a structure-forming salt that stabilizes proteins even when no water is present.

ammonium sulfate can be added to 10% w/v to the cell extract, stirred for an hour, centifuged, and the pellet (which contains ammonium sulfate and some interacting proteins) can be separated from the supernatant. Next, 20% AS can be added to the supernatant and this pellet collected. This can be repeated w 30, 40, and 50%), and each pellet can be analyzed on a gel to determine which products are present. The fraction which contains the protein of interest can be further purifies using chromatography.

Proteins and their mutants can also be digested

and run in 2D to generate a "fingerprint", and unique fragments can be sequences, as was performed by Vernon Ingram, when he identified the amino acid mutation respondible for sickle cell anemia.

18. You are working with a protein that has a pl of 6.25. What buffer pH do you need to use to get this protein to bind to an a) anion exchanger and b) cation exchanger?

anion exchange, give your protein a negative charge, so increase your pH in cation exchange, give your protein a positive charge, so decrease your pH

Affinity chromatography columns make use of...

antibody-antigen interactions, enzymes interacting with substrates, hormones with receptors, proteins with binding partners, or metals binding to specific protein motiffs. The specificity of these interactions is due to complementary surface on the interacting molecules, and usually involves weak interations, although newer techniques sometimes use covalent bonding.

Polycolonal antibodies

are produced by injecting foreign proteins into animals. The animals immune system will see the protein as an invader and generate multiple antibodies to it

CHAOTROPIC SALTs (Urea/Guanidine-HCl)

are used to attempt "re-folding" os inclusion bodies are the opposite of structure forming salts... ***remember inclusion bodied are misfolded protein aggregates these molecules actually decrease the amount of tertiary structure

Several systems make use of the LAC OPERON which is used by...

bacteria to express proteins that break down lactose In the absence of lactose, the lac operon is OFF and the LAC repressor protein is bound to the operator, which PREVENTS E. coli RNA polymerase from transcribing the mRNA required to break down lactose This operon is only ON in the presence of LACTOSE, and therefore can be easily controlled. The LAC OPERON can be used to express recombinant proteins in E coli.

One of the most efficient animal cell systems uses.....

baculovirus to infect insect cells with the gene of interest this systems allows for the expression very large proteins, up to 5000 amino acids, high yields, proper folding, post translational modifications, disulfide bond formation, oligomerization, and simultaneous expression of multiple genes DISADVANTAGE this system is not as straight forward as those in bacteria as it involves eukaryotic cell culture, viral infection, recombination, viral purification, and reinfection. Time and cost are important factors to any research project, and the baculovirus system is more expensive and can take up to two weeks before protein is obtained

8. You wish to separate a 25 and 50kDa protein from one another using SEC. Which size beads should be used? Which would enter the beads and which would elute in the void volume? What would elute in the column volume?

beads with about a 35 kDa cutoff would be ideal for the separating of these two proteins. The 50 kDa protein would elute in the void volume; the original buffer in the column volume

Chemicals such as detergents or organic solvents

can also be used to disrupt the bacterial cell wall or cause osmotic shock, but these can interfere with some downstream techniques such as the Bradford Assay.

Nickel Coated magnetic beads

can also be used to purify his-tagged proteins in a microcentrifuge tube. lysate is mixed with nickel-coates beads, his-tagged proteins bind, and the beads are pulled down in the centrifuge tube, it is then washed and protein is eluted

Antibodies

can also be used to purify proteins, either on a column or on magnetic beads. An antibody can be linked to a bead and a lysate is washed over it The protein of interest can be eluted and further purified

mortar and pestle (blender)

can be used for breaking up eukaryotic cells or organs, would not work for bacterial cells

Gradient gels -separate proteins with a wide range of molecular weights

can be used in which the acrylamid concentration increases, thus porosity decreases, towards the bottom of the gel A gradient gel is a special form of gel in which the degree of porosity is varied continuously from the bottom to the top by creating a gradient in the percentage of the acrylamide

Tissue homogenizer

can be used to break down cells by shearing them or spinning them at a high rpm, but these are usually limited to eukaryotic

Densitometer

can be used to measure the intensity of the bands, which would indicate a relative protein concentration compared to a known marker, this is quantification of protein. While SDSPAGE can provide one with molecular weight and protein concentration by comparison to a marker, these values are only approximations and one would need to further analyze the band to accurately determine

French press

can be used to place cells under VERY HIGH mechanical prssure, up to 40,000 psi, to destroy the cell wall, causing the internal proteins and DNA to be released into the buffer French Press is one of the best disruption techniques, but the resulting lysate can be very viscous/thick due to intact bacterial DNA This can clog purification columns, so sonication and centrifugation are generally performed to break upand remove DNA and other impurities prior to loading the sample on a column. Once lysed, proteins must always be kept on ice or at 4 degreese celsius and purified immediately to avoid protease degredation, oxidation, and aggregation. Multiple cycles of freeze/thawing should be avoided after lysis as ice crystals can destroy proteins. If the protein must be frozen it can be stored in 30% glycerol, but this ay need to be removed via dialysis prior to use.

Enzymes that disrupt the cell membrane, such as lysozyme with bacteria, cellulase with plants, and trypsin with animals,...

can be used, but usually only for small quantties of cells

pH and solubility

can occasionally be used to purify proteins. Proteins are least soluble and often precipitate when the pH of the solution equal their isoelectric point, when the net charge is 0. When dialyzing a protein from one pH to another, a precipitate of unwanted proteins may form that can be filtered or removed via centrifugation. This precipitation can improve the target protein's purity. Inclusion bodies can be found in the pellet.

One of the first techniques developed for protein purification was

centrifugation. After cell lysis, the lysate should be centrifuged in an ultracentrifuge at ~35,000 rpm for 1 hour. This will separate out the bacterial cell wall components, DNA, and other insoluble products such as inclusion bodies into a compact pellet, leaving soluble proteins in the supernatant.

9. 2D gel electrophoresis separate proteins first based on ............., and then based on ................

charge; size

A protease is an enzyme that

cleaves a protein at a specific amino acid sequence. There are dozens of proteases commercially available

The current standard technique for protein purification, used as an alternative to or in conjunction with ammonium sulfate cut-

column chromatography This technique involves loading a protein sample onto a column which contains beds that bind to or separate proteins, and collecting the fractions that drip out the bottom of the column Proteins can be IDENTIFIED USING THE CHROMATOGRAM, which records the absorbance of the solution at 280 nm types of column chromatography as size exclusion, ion exchange, and affinity chromatography

Protein sample treatment, sample loading buffer.. LOADING BUFFER ALSO KNOWN AS "Laemmli buffer"

contains SDS, B-ME, Bromophenol Blue, and Glycerol this is added to the protein sample and the micture is heating to near-boiling temperatures to denature the protein completely. - Bromophenol blue helps in loading protein into wells - The high mobility of the tracking dye assures that it will migrate faster than the proteins - Indicates current is working - When the tracking dye reaches the end of the gel, the electrophoresis is terminated so that the proteins do not run off the bottom of the gel • Glycerol - weigh down the samples by making them more dense than the running buffer and ensures they sink into the wells • Sample may also contain a loading control (actin) that you can stain with antibodies and compare to

Solvent precipitation can be used to

denature, inactivate, or remove unwanted proteins from a sample. Solvents, such as phenol and chloroform, can be useful when purifying proteins that can withstand harsh organic chemicals, but these chemicals often denature proteins or affect downstream experiments. If successful, these organic solvents can greatly improve sample purity, but are usually reserved for DNA and virus purifications.

High Performance Liquid Chromatography, HPLC

developed in 1970s, uses high pressure pumps, up to 100 megapascals, to increase the separation of a sample allows one to separate, identify, and analyze compounds that are present in a sample with outstanding resolution it is used to study drugs, food, cosmetics, environmental samples, forensic samples, chemicals, and for the PURIFICATION OF NUCLEOTIDES AND PEPTIDES. con samples must be able to withstand high pressures, and are not always the best choice for protein purifiation

As the pH increases past a proteins isoelectric point, protons

dissociate, leaving the protein with a net negative charge.

ChIP technology can be used for

genome-wide analysis of transcription factors and epigenetic modifications

ampholytes

having both acidic and basic properties, like amino acids

E.coli as a prokaryotic cell used for protein expression

high levels of expression can express proteins toxic to other systems bacteria is cheap, can be produced by the liter

Resolving gel (10-15% acrylamide)

higher percentage of acrylamide, and performs the separation of the proteins

20. You are interested in knowing if two proteins (40,000 Da and 50,000 Da) you purified interact via dialysis. You can purchase dialysis tubing that has a molecular wight that cuts off 10 kDa, 20 kDa, 30 kDa, ..., up to 200 kDa. Which tubing should you purchase for this experiment? What will remain in the tubing, and what will diffuse into the buffer?

if the two proteins interact, they will have a combined molecular wright of 90 kDa. Therefore, any tubing between50 kDa and 90 kDa (when the two proteins interact), could be used, but 70 kDa would be the best choice as it is halfway between the molecular weights being used. The 90 kDa dimer will remain in the tubing and the unbound 40 and 50 kDa proteins would diffuse into the buffer

Surface Plasmon resonance saturate the system, then measure the time it takes to dissociate

immobilizing protein on a membrane, running its binding partner across to saturate it, and using light to measure this interaction

there are many protein expression systems available to researchers, the simples and most straightforward is this method is fast, EXPENSIVE, and involves a "pure protein that does not nee to undergo purification. ribososome (or cell extract containing ribosomes) and the mRNA of interest this system once perfect may one day replace established expression systems

in vitro (cell free) expression system very low yield, expensive

Denaturation will not break covalent bonds so these modifications will

increase protein's molar weight Modifications include phophorylation(activation) glycosylation (folding/localization) Ubiquitination (destruction)

As the number of modifications increase, the protein's molecular weight

increase, which can be seen on els sometime multiple bands are seen in the same line, indicating different degrees of modification, shown in the gel on the right weak interactions can't be studied

Co-immunoprecipitation Co-IP

involved binding an antibody to a protein on solution, hoping to pull-down other proteins in complex with it to detect new protein-protein interactions

DNA Footprinting

involves adding a protein to DNA and then digesting that DNA and running it on a gel to determine the exact sequence to which the protein binds. The protein will mask the sequence it binds, protecting it from digestion. Normal restriction enzyme digestion will cut the DNA into various-sized piees, but when the protein is bound it protects that region from digestion, and only larger and smaller fragments will be seen on the gel

freezing/thawing

involves freeze/thawing cells for about 3 cycles since the ice crystals will disrupt cell membranes.

Immunoprecipitation IP

involves precipitating an antigen out of solution using an antibody as a purification step

Electrophoretic mobility shift assay (EMSA), involves running DNA or RNA on a gel,

involves running DNA/RNA on a gel, followed by DNA with a potential binding partner and looking for a shift on the gel, which indicates PROTEIN-DNA INTERACTION. The protein can be identified by using an antibody and looking for further shifting.

Chromatography

involves the separation of a substance by using a stationary phase, such as beads, and mobile phase, sample and buffer. Can be performed as liquids, gases, and under high or low pressures.

Ammonium Sulfate Cut (salting out)

is a controlled precipitation where proteins are gently removed from the buffer and stabilized by the positive charges of the ammonium and negative of the sulfate ALLOWS ONE TO PURIFY PROTEIN FROM CELLS OR ORGANS CAN LEAD TO THE ISOLATION OF NEW PROTEINS. IT IS INEXPENSIVE AND DOES NOT REQUIRE ANY CLONING OR PCR PROTEINS WILL BE FROM THEIR NATURAL SOURCE AND THEREFORE CONTAIN ALL APPROPRIATE POST-TRANSLATIONAL MODIFICATIONS The disadvantage of this technique is that it can be difficult to obtain large amount of pure proteins, as many cells or organs are required as starting materials These proteins may also aggregate with their natural binding partners, this make PURIFICATION MORE DIFFICULT Cannot be purified via affinity chromatography, which is the best protein purification technique available. Ammonium Sulfate Cuts are used if chromatography was not successful or if one wants to clean up a lysate prior to adding it in a column.

Acrylamide (toxic)

is a small molecule that will polymerize into chains under the right conditions, and the porosity of the polymerized polyacrylamide gel can be controlled

TEMED (tetramethylethylenediamine) induces polymerization in gel

is a tertiary amine that reacts with these radicals to form TEMED free radicals, which in turn react with acrylamide to induce polymerization

Ammonium Perfulsulfate (APS) -catalyst for substates acylamid and BIS

is necessary to catalyze the reaction between acrylamide and BIS (-O3S-O-O-SO3-), feadily forms unstable SO4- radicals

Reserve-Phage chromatography

is similar to HIC, but is generally used on samll molecules such as peptides, which are denatured and loaded onto a column containing long-chained hydrocarbons. • Performed under denaturing conditions - separation based on hydrophobicity • Elution of peptides with acetonitrile or methanol in the order of their increasing hydrophobicity

Gel Electrophoresis (travel based on charge) can be used to separate and analyze both DNA and proteins

is the separation of charged molecules through a gel matrix in order to sort them by size and or charge

protocol for both IP and CO-IP

is to add an agarose bead with a bound antibody to a cell extract In immunoprecipitation, the antibody will bind to the protein of interest and it can be spun and washed. The protein can then be run on SDS-PAGE, mass spec, or a western blotfor identification For Co-IP is similar except that the protein bound to the antibody will hopefully interact with its binding partners in the cell extract, and you can pull them out of solution together and analyze the sample Co-IP very useful for identifying new protein-protein interactions

A SECONDARY ANTIBODY OPTION OPTION

is to inject protein or epitope into a mouse, rabbit, goat, or other animal and purify it, but this REQUIRES A LOT OF TIME AND EXPERTISE this secondary antibody can be covalently coupled to an enzyme that catalyzes a chromogenic reaction, such as horseradish peroxide (HRP) or alkaline phosphatase The prsence of the secondary antibody can be assayed by immersing the membrane into the substrate of the coupled enzyme which CAUSES A COLOR CHANGE OR FLUORESCENCE

Far Western Blot, variation of western blot allows one to identify protein-protein interactions RADIOLABELED bait protein probe instead of a primary and secondary antibodies

it is essentially a western blot with a few minor differences, such as a native gel, and a RADIOLABELED bait protein probe instead of a primary and secondary antibodies IF THE BAIT INTERACTS WITH ANY PROTEINS ON THE GEL, IT WILL BIND AND CAN BE DETECTED WITH AUTORADIOGRAPHY

Yeast cells and plant cells as eukaryotic cells for expression of their recombinant proteins

large amount of protein, high yield, very difficult, tedious, and timely cells divide more slowly than prokaryotic systems efficient cleavage of signal peptides, expression multiple genes more difficult than bacterial system

Detergents

lower expression temperatures and/ore detergents, such as Triton X-100m Sarkosyl, and CHAPS, can be used to resolubize the sample, and are useful when working with membrane proteins, which tend to aggregate due to an exposed hydrophobic region that inserts into the cell's phospholipid bilayer.

SDS-PAGE Applications molar weight determination Used to track proteins used in conjugation with chromatography to test fractions and confirm presence of protein of interest covalent modifications of proteins can be studied on SDS-PAGE

many applications/important in molec bio it can also be used to SIMPLY DETERMINE A PROTEIN'S MOLAR WEIGHT, or as a part of a purification to track expression and locate the protein of interest. It can verify what you see on a chromatogram SDS-PAGE is also used as part of larger experiments involving protein modifications and protein mutation analysis

Isothermal titration calorimetry

measures heat change upon binding when one protein is titrated into a tube containing a binding partner, such as a peptide and a protein that interact. When the two bind a small amount of heat will be given off or absorved depending on whether the interaction is exothermic or endothermic

This LEAKY uncontrolled expression may result in

misfolded protein since many proteins are expressed at lower temperatures, or the protein may kill the bacteria if it is toxic to them.

In some cases, the polyclonal antibody may be more useful than the...

monoclonal antibody for immunohistochemical studies

monoclonal and polyclonal

monoclonal high specificity and low affinity polyclonal low specificity and high affinity

Recombinant DNA technology

most labs working with proteins begins at DNA level with PCR, cloning, restriction enzymes, and bacteria, and the end result of such manipulations is the insertion of human gene of interest into a bacterial plasmid, also called vector or construct this vector is called recombinant dna technology since it contains dna from various species and is not natural. This plasmid-gene hybrid can be transformed into bacteria where the gene can be expressed

Electrophoresis

movement of charged molecules toward an electrode of the opposite charge - If an electric field is placed across a solution containing ions a current will develop and charged molecules will separate - Anions move toward the anode (+ electrode) - Cations move toward the cathode (- electrode)

Anion Exchange Chromatography Columns

negatively charge proteins bind to POSITIVE beads Chloride ions for the anion exchangers (Cl-)

beads in cation exchange chromatography are

negatively charged.

To Perform a Western Blot

one must first run an SDS-PAGE of the sample proteins. Afterwards, the gel is removed from the gal sandwich and placed flat onto a nitrocellulose or PVDF (polyvinylidene difluoride) membrane. Nitrocellulose is less expensive PVDF has a higher binding capacity, although it needs to be activates in METHANOL The gel/membrane sandwich is placed between two pieces of filter paper soaked in buffer to allow for the electric current to pass through the complex. This setup is placed in an electric current perpendicular to the gel, which causes the proteins to migrate out of the gel onto the membrane to which they adhere THIS PROCEDURE IS THE WESTERN TRANSFER After western transfer, the gel is discarded, THE MEMBRANE, which now has BOUND PROTEINS, still has many free binding sites available. If an antibody, which is a protein, was added to the membrane at this point, it would bind all over. The membrane is THEREFORE BLOCKED by placing it in milk.

Overall all net charge of protein depends on...

pH of surrounding area

Native Gels can be used to analyze the degree of

phosphorylation on serines, threonines, and tyrosines, which are too small of a mW change to see on SDS PAGE Native gels can also be used to study glycosylation at various amino acids. one can determine proteins OLIGOMERIC STATE, they will migrate differently on native gels. SDS SEPARATES HETERODIMERS AS TWO SEPARATE BANDS WHEREAS IN NATIVE GEL IT IS READ AS ITS OWN BAND

Cation Exchange Chromatography Columns

positively charged proteins bind to NEGATIVE beads - Metal ions for the cation exchangers (Na+)

13. What is the charge of the beads in anion exchange chromatography?

positively-charged beads

Cells can also be immediately lysed

producing a lysate or homogenate it is important that lysis takes place in a buffer in which the protein is stable, and pH and salt concentration ms be taken into consideration.

Protease digestion is another technique that allows for

protein analysis, including domain analysis, conformation and activation analysis, and protein fingerprinting.

For these purification methods...

proteins can be identified/later location thourgh SDS-PAGE and wester blotting

tandem affinity purification

proteins in this method are designed to contain multiple tags to improve purification by adding STEPTAVIDIN-BINDING PEPTIDE and CALMODILIN BINDING PEPTIDE, and then passing them over both affinity columns yield and exceptionally pure protein

As the pH decreases past a protein's isoelectric point,

proteins will be protonated and have a net positive charge

antibodies can also be used to

purify proteins on a bead or column also useful in detecting dna-protein interaction firectly on chromosomes, which will be discussed in the next chapter

4-6

refer to charts in book

8. Which has a higher % of acrylamide? Stacking Gel // Resolving Gel

resolving gel

differential scanning calorimetry

scans a protein across different temperatures and measures the results. This can be useful when comparing a protein vs a mutant, as the two might behave differently at different temperatures.

ELISA, (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. -reliable, and only take minutes to perform -SDS not used, samples are placed in small wells -color change is proportional to the amount of antibody -it provides NO INFORMATION ON THE MOLECULAR WEIGHT OF AN ANTIGEN -An antibody in ELISA could bind nonspecifically and produce a false positive

similar to western, ELISA (enzyme-linked immunosorbent assay) allows for the DETECTION of an antigen or antibody in a sample it is often used as a quick medical diagnostic tool to detect the presence of certain protein markers or viruses in a patient, in industry to check sample quality, and in pregnancy tests ELISA is nearly identical to Western Blot EXCEPT that an SDS-PAGE gel is not ran. samples are placed in small wells and assayed, often many at a time If a reaction occurs, the well will change colors, indicating that an antibody bound to an antigen. While very sensitive, this technique is at a disadvatage over the western blot bc it provides NO INFORMATION ON THE MOLECULAR WEIGHT OF AN ANTIGEN An antibody in ELISA could bind nonspecifically and produce a false positive

Mammalian cells can be used for expressing recombinant proteins....

slow, expensive, low yield.. these cells allow exact protein with all proper modifications.

11. If you perform size exclusion chromatography on a lysate, the fastest-moving proteins in the column will be on the (slowest/fastest)-moving proteins on an SDS-Page gel.

slowest

10. Which runs fastest on an SDS PAGE gel Large Proteins // Small Proteins

small proteins

Metal chelate chromatography technique produces very pure, unusable proteins after a single step, often followed with IEX and SEC to purify sample further.

some proteins use histidines to bind to metals, such as in the case of carbonic anhydrase's active site containing three histidines that coordinate zinc, an essential cofactor to catalysis This led to Metal chelate Chromatography Nickel or Cobalt, lower affinity also reduce contaminant binding, are loaded onto a column and proteins with HIS tags are passed over column. Gene of interest is genetically modified to encody a polyhistidine tag, usually six-8 histidines in length at either N or C terminus. This tag can be part of a plasmid or encoded directly into a PCR primer. - Nickel column • Nickel (and other metals such as cobalt) bind 6-8 histadines • Elute with imidazole

Sonication

sounds waves vane be used to break up cell walls and cell membrances, but only in short bursts and on ice to prevent overheatin of sample or breakign the beaker

FINALLY, A GREAT OPTION IS TO USE ANTIBODIES SPECIFIC TO A ..

specific to a TAG ON A PROTEIN SUCH AS GST, MBP, A HIS-TAG, FLAD-TAG, OR GFP.

Protease Inhibitors

such as PMSF, EDTA, and EGTA are often addid to this buffer to prevent degredation

5. You load a mixture of 15kDa, 25kDa, and 50kDa proteins onto a size exclusion column. Which will be collected in the first fractions? Which in the last fractions?

the 50kDa proteins will be the first to be collected because they are the largest, then the 15 kDa will be collected last, they are the smalled

Gene Fusion Technique -variation of affinity chromatography that is helpful when purifying recombinant species

the gene encoding the protein of interest is fused to a second gene encoding a protein that is easily purified by affinity chromatography. This second protein is called the "tag" Gene Fusion - GST column • Glutathione binds glutathione-S-transferase • Elute with glutathione - MBP column • Amylose binds MBP • Elute with maltose

Upon IPTG indution of the E. coli expression system that contain this plasmid,

the lac repressor is removed from the operator, E. coli RNA polymerase can transcribe the gene of interest. This expression system works but can be LEAKY, meaning it is more likely to have low levels of protein expession, even in the OFF state.

The most important step to chromatography is identifying the fractions that contain

the protein of interest

The difference between the buffer pH and the protein's pI affects..

the rate at which the protein will travel through the column Proteins in a buffer with a pH very close to their pI may not completely bind to the column, but rather move very slowly through the column compared to neutral proteins Generally, a pH of 1 unit or slightly greater away from the pI is chosen to ensure that proteins will completely bind to the column.

3. What would be the result of forgetting the blocking step in a western blot?

the result of forgetting the blocking step in a western block would result in the primary antibody, protein, would bind all over the membrane, and not just to the protein of interest the block step ensures the antibody binds specifically to the protein of interest

Bacterial/Yeast Two Hybrid to Study Protein Interfaces

the system can be set up such that a bait and binding domain are generated on one plasmid, and an entire cDNA library with potential binding partner genes next to the activating domain on a second plasmid. It allows for screening of thousand of potential binding partners at a time If a color change is present, the binding partner can be identified and further studied

chaotropic salts "refolding proteins"

these molecules actually decrease the amount of tertiary structure in a protein, reducing it to linear state. The chaotrophic molecules can then be slowly removed with a gradient on a column/dialysis, and the denatured proteins will HOPEFULLY REFOLD PROPERLY Not a desirable purification method its success is limited to and used as a LAST RESORT

Liquid chromatographies (fast protein liquid chromatography FPLC) (low pressure liquid chromatography LPLC)

these types of chromatography are types of liquid chromatography • Used to separate metal ions and organic compounds in solution • The mobile phase is a solvent and the stationary phase is a solid, gel, or resin

1. Researchers generally use recombinant DNA technology to express proteins. Why can't proteins be synthesized from amino acids via organic chemistry?

this technique is currently limited to about 50-70 amino acids

18. What is the purpose of dialysis?

to remove salts from a buffer, and to prepare that buffer for future purifications and storage

Standard curve

to simplify molecular weight analysis, a standard curve can be determined using the markers, which have known molecular weights, on a gel with a computer software. A protein band can be selected and the computer will determine the molecular weight based on this standard curve

One advantage of ELISA is that it can be

used in plated which screen dozens of samples at once

3D Gel Electrophoresis

used to increase resolution even further Native gel -> IEF -> SDS-PAGE

Protein purification

useful technique for isolating a specific protein from a lysate either preparative or analytical purposes. Researchers may wish to purify an enzyme for further studies, identify and quantify a protein, analyze protein-protein and protein-DNA interactions, or use it in structural studies Proteins must be purified from the cell lysate. The process involves increasing the concentration of the desire protein relative to all other unwanted proteins. Every step of a purification process will result in loss of protein, both wanted and unwanted, but the goal is to lose less of the protein of interest than the unwanted proteins.

Without the stacking gel , the protein bands on a gel would be

very wide and likely unreadable

Mass spectrometry protein digested, molar weight compared in sample fragments. most accurate molar weight. - Digest protein - Analyze fragments - Repeat with different enzymes - Rebuild protein

where a protein is digested, and the molecular weights are analyzed and compared to known fragments. The protein's sequence can be determined by overlapping the fragments

SDS can be used to study mutations, which can help to determine...

which amino acids are involved in protein activity, protein modification, protein function, and protein interactions

Thin Layer Chromatography (TLC)

• A simple and rapid method to monitor the extent of a reaction or to check the PURITY OF ORGANIC COMPOUNDS • The mobile phase is a solvent and the stationary phase is a solid adsorbent (such as paper, cellulose, or silica)

Principles of AC (from powerpoint)

• Approach: chemically couple a small molecule or protein to an inert material • Dextran, polyacrylamide, or agarose provides a stable open gel structure, which large proteins can penetrate, and a large surface area to which the immobilized group (affinity ligand) can be linked • These stationary matrixes can be formed into beads with a large number of reactive groups • Affinity ligand is commonly linked to free hydroxyl groups on the matrix Elution • After washing the noninteracting molecules away, the target molecules are eluted • Highly specific elution of the desired macromolecule from the stationary phase is achieved by adding a gradient of a competing agent to the buffer, which is usually the same molecule that was immobilized on the column • Used to purify specific biological macromolecules: - Enzyme bound to substrate -> Mobilized substrate • GST/GSH • MBP/Maltose - Antibody bound to antigen -> Mobilized antigen - Hormone bound to receptor -> Mobilized receptor - Metal bound to histidines -> Histidine ring

Chromatography Components

• Chromatography column • Gel matrix • Buffers • Gradient mixer • Peristaltic pump • Conductivity meter • UV detector • Fraction collector

Two-Dimensional Electrophoresis

• Combination of IEF and SDS-PAGE • A tube IEF gel is run, and the resulting tube gel is then placed horizontally across the top of the an SDS-PAGE gel • The proteins are separated by their pIs in the first (IEF) dimension and according to their molecular mass in the second (SDS-PAGE) dimension • Since both of these techniques are of very high resolution, the resolution obtained when both are combined is outstanding resolution

Preparative Applications of SEC

• Compatible with physiological conditions (pH, salts) and can be used with most protein samples • Large sample protein separation - Proteins (or other macromolecules) of different molecular weight - Monomers from dimers, trimers, tetramers, etc. - Separate monomers from aggregated proteins or degraded products, which often form during protein purification - Since the salts/buffer from the sample take the longest path, the proteins fall into the buffer that the column is equilibrated in. Therefore SEC is often incorporated as a final polishing step to remove aggregates and act as buffer exchange mechanism into the final solution

Detection and Analysis

• Detection - Visible compound production (TMB) - Chemiluminescence - Direct Infrared fluorescence (picograms) - Autoradiography (picograms)

Oligomer Determination

• Example - signal transduction protein NRII (nitrogen regulator) - NRII homodimer is two 37.5 kDa wild-type NRII subunits - Hybrid heterodimer is one 77.5 kDa MBP-NRII subunit and one 37.5 kDa NRII subunit - Fusion protein homodimer is two 77.5 kDa MBP fused to NRII subunits - After excision of the different dimer bands, the enzyme was denatured and fractionated by SDS-PAGE to observe the number and size of the subunits

Electrophoresis (Sieving) vs. Gel Filtration Chromatography

• Gel filtration: molecules are excluded from the interiors of the gel beads according to their size, so that the largest molecules elute first • Gel electrophoresis: the gel matrix is a mesh-like substances rather than a bead, and therefore, it acts as a sieve, where frictional forces decrease the mobility of larger molecules more than that of smaller molecules • Therefore, large molecules travel more slowly than small molecules

IEF application

• IEF is useful for determining the pI of proteins • It can detect small changes in proteins such as covalent modifications or proteolysis. • However, it requires special equipment (capillary gels and ampholytes) which can be EXPENSIVE, and it is therefore NOT THAT COMMON of a technique in the laboratory • In combination with SDS-PAGE in the two-dimensional electrophoresis method, IEF has played an important role in the identification of numerous proteins

Size Exclusion Chromatography, SEC GENTLE METHOD FOR PURIFYING NUCLEIC ACIDS, SUGARS, AND PROTEINS. SEC generally final step is to remove aggregates and act as buffer exchange SEC is both analytical and preparative

• Jerker Porath and Per Flodin (Sweden) cross-linked dextran and showed proteins could be separated by size • SEC - also called gel filtration or gel permeation chromatography • The mobile phase is a solvent and the stationary phase is a packing of porous particles • SEC is a mild method for purifying nucleic acids, sugars, and proteins: - Separating molecules groups based on size (MW) - Desalting (buffer exchange) - Removing ammonium sulfate after a precipitation • Very Reproducible • The sample is applied in a narrow band at the top of the column and then washed through the column across the mobile phase • Disadvantage: band broadening (inject <10 ml)

Quantification of Protein Bands

• Markers • Standard curve • Stained gels can be scanned in a DENSITOMETER to quantify the relative amounts of the various proteins bands (concentration) • Individual proteins bind to the dye distinctly, and in the absence of specific knowledge on the binding of each protein to the dye, quantification of stained bands is ONLY APPROXIMATE (like DC protein assay, Bradford assay, gel filtration Mw determination)

Selecting a Column

• Prepacked, dry powder • Size exclusion • IEX - Anion - Cation • Affinity - Nickel - GST - Maltose • Columns have a maximum load, and longer/larger columns will hold more protein and offer better resolution

Coomassie-Stained Slab SDS-PAGE Gel

• Protein from SEC column • An aliquot from every third fraction was run, from left to right on the gel • A prominent band is observed samples 4-9

Protein Microarray

• Protein-protein interactions, kinase/substrate interactions, transcription factor screening • Drug screening • Screening for immunity to certain proteins or infections

Stacking Gel and Buffers

• Proteins be applied to the gel in very small volumes to obtain good separation • A stacking gel is cast directly on top of the larger resolving gel to allow for larger volumes to be loaded • Because the stacking gel is very porous, there is little difference in the mobility of proteins of different size as they migrate through the stacking gel • The proteins are all concentrated into a narrow band at the top of the resolving gel • The stacking gel is polymerized with a lower percentage of acrylamide to ensure high porosity and the resolving gel contains a higher percentage of acrylamid

Detergent and b-Mercaptoethanol reducing agents, reduce cross-links are both components of SAMPLE LOADING BUFFER

• SDS disrupts most of the interactions that hold proteins together, including oligomers, resulting in monomeric polypeptide chains • However, disulfide cross-links between polypeptide chains are not broken by heat or SDS, and thus polypeptide chains so linked will remain covalently bound • These cross-links are eliminated using the reducing agent b-mercaptoethanol which is added to the sample along with SDS • Dithiothreitol (DTT) is less volatile and more effective, but more expensive • Some proteins are very resistant to binding SDS and denaturation

Excised Dimer Analysis on SDS-PAGE

• SDS-PAGE analysis of the dimers excised from the gel shown on the previous slide

Western Blot Procedure

• SDS-PAGE of the sample proteins • Transfer: - The gel is removed from the gel sandwich and placed flat onto a nitrocellulose or PVDF membrane - A set of absorbant pads is used to support the gel and membrane as a sandwich, and the assembly is held in a supporting clamp - An electric field perpendicular to the sandwich forces the proteins to migrate out of the gel and onto the membrane to which they adhere http://www.anaspec.com/images/2014_ 10/iDBlotPrinciple.jpg • Block: - Because the membrane has free sites remaining, it is coated with a mixture of nonspecific proteins to block these free sites (dry milk)

Size Exclusion Chromatography Logistics

• Separation based on size and shape • Contains porous beads ~100 mm in diameter • Beads have an "exclusion size", in the QBM lab we use Sephadex G-50 or G-75 beads • Total liquid volume >95% of the packed column • Separate molecules ~2X difference in Mw • Very large molecules completely excluded (void volume) • Salts/buffer comes off last (column volume) he Gel Matrix • The AMOUNT of cross-linking determines the average PORE SIZE of a gel • Different degrees of cross-linking give different pore sizes and different molecular sieving or separation ranges • Three types of polymers, each cross-linked to varying degrees depending on experiment - Dextran (Sephadex) beads - Polyacrylamide - Agarose

Reversible Uridylylation of PII

• Since the PII protein is a trimer of identical subunits and uridylylation occurs at unique sites within each subunit, there are four possible states corresponding to 0, 1, 2, or 3 uridylyl groups per trimer • Lanes 1-4 show a time course of uridylylation of PII • Undetectable by SDS-PAGE

Stokes Radius

• Stokes radius is the effective radius a molecule has as it tumbles rapidly in solution (i.e. the radius of a hard sphere that diffuses at the same rate as the molecule) • A long extended molecule has a larger stokes radius than a compact molecule of the same molecular mass

Acrylamide Pore Size

• The average size of pores can be controlled by varying: - The amount of monomer (acrylamide) used - The degree of cross-linking • Higher degrees of cross-linking results in narrower pores • The degree of cross-linking is generally kept constant, and the percentage of monomer is varied to make gels of different porosity • The pore sizes of the acrylamide gel affect the migration rate of protein: - Small proteins will run through the gel quickly because they can easily fit through the pores - Larger proteins migrate more slowly as they make their way through the gel - Only proteins of the correct size can enter the pores, and some proteins are so large that they may not be able to enter the gel at all

Polyacrylamide Gels

• The porosity of the polymerized polyacrylamide gel may be controlled by varying the percentage of acrylamide and/or the degree of cross-linking of the acrylamide chains • Polyacrylamide gels are formed by the polymerization of acrylamide

Western Blot Procedure

• The secondary antibody is usually coupled covalently to an enzyme that catalyzes a chromogenic reaction - Horseradish peroxidase (HRP) - Alkaline phosphatase • The presence of the secondary antibody can be assayed by adding substrate of the coupled enzyme to the membrane, which causes a color change or fluorescence • Why two steps? Primary antibodies conjugated to enzymes are available - Versatility and adaptability to new proteins - Signal amplification (polyclonal vs. monoclonal)

Electrophoresis of Proteins

• The separation of proteins gel electrophoresis is influenced by - The charge of the proteins at the buffer pH - The size of the proteins and the frictional resistance as they migrate in the electric field • If these two characteristics balance each other out, proteins of different charge and size can migrate at the same rate in an electrophoresis experiment

Visualization of Protein Bands

• Tightly binding dyes - requires soaking gel in a solution - Coomassie brilliant blue R-250 • A dye closely related to that used in the Bradford assay for total protein concentration • Binds strongly to proteins (aromatics, arginine, histidine) • Destain with methanol • Not very sensitive - Silver staining • Silver atoms bind very tightly to proteins and produce black or purple bands (as in photography) • 50-100X more sensitive • More expensive, longer to perform • Toxic • Fluorescence - 2,2,2-Trichloroethanol (TCE) - reacts with tryptophan under UV - Can be used downstream (i.e. western blotting) - The most common groundwater contaminant in the U.S. • Radioisotope detection (autoradiography) - X-ray film - 35S in methionine, 32P, 33P (X-ray) - 14C, 3H (fluorophores) • Antibody detection (Western Blotting)

Buffers - Tris-Glycine

• Tris - trishydroxymethylaminomethane (2-amino-2-hydroxymethyl-1,3-propanediol) and glycine • The upper and lower reservoir buffer contains Tris pH 8.8 with glycine as the counterion • The stacking gel (in Tris pH 6.8) contains the protein mixture and tracking dye • The pH difference generates an ion gradient via glycine, Cl-, which carry the current, and assists in the stacking process

Field Effects in SDS-PAGE include

• Uneven electric field • Overheating • Incorrect polymerization

Dimeric Forms of NRII of E. Coli

• Wild-type NRII is a homodimer of two 37.5 kDa subunits • MBP-NRII is a homodimer of two 77.5 kDa subunits, consisting of MBP fused to NRII • Hybrid dimer is heterodimer with one NRII and one MBP-NRII subunit, midway between the wild type and the MBP-NRII dimer