CHEM 153L FINAL CONCEPTS
What do you need to consider about laboratory safety when working in a biochem lab? How do you prepare yourself for lab experiments before you enter the lab and what do you do inside the lab to stay safe and keep the environment safe? What do you do inside the lab, when an accident happens?
- To stay safe in lab, DO NOT wear gloves outside of labs, DO NOT touch telephones with gloves, DO NOT eat or drink - In case of accident, call 911 from lab phone. Eyewash, shower, fire extinguisher, etc.
What are the various ways to remove your protein of interest from the column (elution)?
- change in pH - increase in salt concentration - addition of competitor of ligand - addition of competitor of target
What is a trial overexpression? Why is a trial overexpression advised?
- due to different functions, different proteins are not all overexpressed as easily as other. - for each new POI you need to find optimal growth conditions for cell to produce most POI (temp, concentrations, duration of induction)
What is the purpose of immunoblotting?
- monitor subtle changes in expression of specific proteins in different samples (even when expression levels are not high, changes are visible)
Remember why we needed to transfer proteins onto the membrane. How do you get proteins to move from gel to membrane without losing their position relative to each other?
- pores in the gel are too small to allow for efficient diffusion of antibody to access all proteins. - horizontal electric field to keep the protein separation by molecular weight established in the protein gel, but to remove proteins from gel matrix and bind to the surface of the nitrocellulose membrane to allow even access of antibodies to all proteins on the membrane
Why is protein overexpression useful?
- produce POI in high amounts, often with tag, and often in cells easy to grow
Why is it advisable to use a stacking gel in SDS PAGE? What would we see if a stacking gel was not used? Would proteins migrate?
- proteins are randomly dispersed in the protein sample buffer and will be randomly distributed in a relatively high, but thin well in the gel (since gel is very thin). - stacking effect helps to prevent those proteins from smearing in the separating gel by starting the proteins all at the same time before going into the resolving gel. - without stacking gel, we would see proteins smearing in our gel.
Why does the molecular weight of proteins not affect their speed of migration in the stacking gel but in the resolving gel?
- same charge/mass ratio - large pores - all proteins can be same speed (except when they are pushed faster due to high local voltage from glycine zwitterions (which is not related to molecular weight, but only due to their random position in the well))
What experimental steps did you do in the laboratory to produce biofuels?
- use cells that harbor the plasmids with the required gene sequences - inoculate fresh culture - let grow to exponential phase - induce with IPTG - incubate for 24 hours
Differentiate what we mean by 0th order and 1st order reaction conditions? What would you do in the lab to set up one or the other?
0th order: high substrate concentration used so the supply of substrate is no longer limiting. product formation rate solely depends on enzyme concentration. in this range of substrate concentration you will not see a change in activity rate with increasing or decreasing substrate concentrations (if you decrease too much, you will reach first order conditions where substrate availability is limiting the enzymatic rate).
ENZYME ASSAY: Write down all experimental steps (like your flowchart) and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
1. Add enzyme diluent to tube with unknown pH buffer 2. Read this in spectrophotometer at 340 nm quickly (this is blank) 3. Add 1/10 diluted enzyme to 2nd tube with unknown pH buffer. Read in spectrophotometer. - this is to find optimum pH buffer to measure enzyme activity 4. Prepare tubes with various ethanol concentrations + NAD+ + EtOH + buffer +water 5. Add diluted enzyme to each and measure absorbance 6. Determine product formation rate with Beer's Law then plot to make Michaelis-Menten graph for Km and Vmax
Describe the various steps of an affinity chromatography and their respective purpose
1. Equilibration of ligand (add low concentration of imidazole to the column): reserve binding spot for His tag 2. Binding of protein of interest to the column while non-specific proteins pass column and will be caught in flow through 3. Washing of all proteins that bound non-specifically): slightly increase the imidazole concentration in the solution to reduce non-specific binding and therefore to wash off other proteins 4. Elution: drastically increase the imidazole concentration to elute all the protein of interest from the column 5. Re-equilibration of ligand: preparation of future use
How do you stain all proteins in the gel?
1. Incubate gel in Coomassie Solution 2. Remove all unbound Coomassie dye by destaining RESULT: only proteins stay stained (blue bands)
How can you determine Ki experimentally? (Hint: look at Lab 6 worksheet)
1. Perform enzyme assays under various substrate concentrations with and without inhibitor present 2. Find the ratio of Km apparent over Km=alpha 3. Use alpha = 1 + inhibitor concentration/Ki 4. Plug in inhibitor concentration and solve for KI.
BRADFORD ASSAY: Write down all experimental steps (like your flowchart) and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
1. Prepare various known concentrations of known protein standard in dilutions 2. Prepare various dilutions of unknown protein solution 3. Add known and unknown protein solution + Bradford reagent into new tubes 4. Pipet in triplicates into 96 well plate 5. Use plate reader at 595 nm
PROTEIN EXPRESSION: Write down all experimental steps and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
1. Put LB medium in flask 2. Add ampicillin to LB medium in flask 3. Get optical density of overnight culture - this is to determine how much volume to add to flask to normalize. 4. Add overnight culture to flask 5. Short incubation of 30 minutes at 37C 6. Remove 2 samples for 'time 0' 7. Add IPTG to remaining culture, place in incubation for 1 hour 8. Freeze centrifuged supernatant of 1 tube for SDS page, for the 2nd tube measure its OD 9. Repeat same steps for Time 1 and Time 2
Besides the histidine tag and Ni+2, name two other affinity pairs (ligand/target) that can be used for affinity chromatography
1. antibody and antigen 2. substrate and enzyme/inhibitor 3. glutathione-s-transferase and glutathione
Describe two advantages of biofuels as compared to fossil fuels
1. biofuels are renewable while fossil fuels are limited. 2. biofuels do not add to CO2 in atmosphere since plants capture atmospheric carbon dioxide and we release them in biofuels (net zero)
ISOBUTANOL PRODUCTION IN E.COLI: Write down all experimental steps (like your flowchart) and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
1. receive innoculated cultures and control (SA542) 2. dispense 1xM9 medium in 3 tubes with ampicillin and kanamycin 3. dipsense 1xM9 medium in another tube 4. add overnight cultures and incubate 5. add IPTG after 2 hours then incubate overnight - to induce gene expression 6. Prepare various tubes with known isobutanol concentrations to generate standard curve 7. Prepare various tubes with unknown isobutanol concentrations 8. Add ADH2 to all assays then incubate in dark then measure absorbance at 570 nm
AFFINITY CHROMATOGRAPHY: . Write down all experimental steps (like your flowchart) and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
1. receive pellet that was induced with IPTG 2. resuspend pellet in Bugbuster bacterial lysing reagent 3. centrifuge and keep supernatant - separate soluble + insoluble 4. equilibrate Ni-NTA spin column by adding NP-10 then discarding 5. load lysate supernatant in column and centrifuge 6. repeat three times until lysate is gone 7. wash column with NPI-20 and centrifuge repeatedly - remove as many non-specifically bound proteins as possible 8. Add NPI-500 buffer and centrifuge for eluates
ISOBUTANOL PRODUCTION IN E.COLI: Remember the purpose of using relevant chemicals for this method (those that we covered in lecture). Which results would you expect if the incorrect molecule was used instead.
1xM9 medium: IPTG: ADH2:
When you set up this reaction in the lab, what else do you need to add? Why?
50 mM pH buffer to find the optimum pH for the enzyme
You are performing an affinity chromatography purification like in laboratory 4. After collecting all the fractions, you take small aliquots of each to run them by SDS PAGE and check if you purified the protein efficiently by Coomassie staining. Explain shortly what might have happened in below scenarios Soluble lysate (after centrifugation to remove insoluble material) and flow through contain many protein bands (including a thick band in the size range of your protein of interest), there are a couple of thin bands in wash step 1, no bands in wash steps 2 and 3, a thick band in eluate 1 and eluate 2.
A thick band in eluate 1 and 2 shows us that the purification process worked and it is reasonable that there are a couple thin bands in wash steps because it is possible to wash off some POIs. Yet the thick bands in flow through shows that there are many POIs in flow through and indicates that they cannot bind to the column. When we combine all these, we then can reach the conclusion that there are too many proteins in the supernatant and therefore, not all POIs can bind to the column and they come out in the flow through. A possible solution is to have a larger column, or dilute supernatant, or split it into more fractions.
What molecules are not involved in gel formation? What are their respective roles?
Acrylamide: monomer that is polymerized to form gel matrix (and thus pores) Bisacrylamide: crosslinks linear polyacrylamide strands to each other and thereby strengthens the gel Ammonium persulfate and TEMED: start radical reaction that forms polymer
Is EDTA specifically inhibiting alcohol dehydrogenase OR will other enzymes be inhibited by EDTA? If yes, which ones?
All enzymes that need divalent metal cations for their function will be inhibited in the presence of EDTA as it effectively reduces the availability of the metal cation. DNase, RNase, and metalloprotease
What is alpha with respect to competitive inhibition?
Alpha indicates the degree of inhibition For competitive inhibitor, it is the ratio of Km apparent over Km
Indicate how you can measure the enzyme reaction (i.e. which molecule are you actually measuring in the spectrophotometer). Keep in mind, if the absorbance is expected to rise or fall in the reaction you wrote above.
Amount of NAD+ reduced to NADH over time is measured as an increase in absorbance at 340 nm under initial rate conditions
Which pH conditions would you set for Coomassie staining?
Anything above 2, so that the equilibrium is pushed towards the blue form Coomassie staining will remove all unbound dye molecules (unlike Bradford Assay)
How can you adjust your analysis to generate a longer linear correlation?
Assess background absorbance of red dye by measuring absorbance in every sample for red dye from (470 nm) and blue form (595 nm). Then take the ratio of 595/470 absorbance values. This accounts for changing background
BRADFORD ASSAY: Remember the purpose of using relevant chemicals for this method (those that we covered in lecture). Which results would you expect if the incorrect molecule was used instead.
Bradford Reagent/Coomassie Blue dye: binds to all proteins via basic and aromatic amino acid side chains and protein backbone
Describe the properties of Coomassie Brilliant Blue and why these properties allow us to determine protein concentration. Recognize the Coomassie Brilliant Blue molecule
CBB changes colors depending on pH (blue when bound to proteins, red and green when unbound) Red = +1 charge Green = 0 charge Blue = -1 charge
Which detection method did you use in the course?
Colorimetric detection with nitroblue tetrazolium and 5-bromo-4-chloro-indolylphosphate. The phosphate is cleaved off by alkaline phosphatase and only then can both molecules react to form a color on the membrane (this should only happen where secondary antibody with conjugated alkaline phosphatase is present)
What are the options of visualizing your POI on the membrane?
Colorimetric, chemiluminescent, and fluorescent detection methods
What are loading controls?
Coomassie stained gel (or part of gel) OR immunoblot against housekeeping gene
Could you answer the above question, if you were given the specific enzyme activity instead of the kcat (and no other change or additional info)?
Could you answer the above question, if you were given the specific enzyme activity instead of the kcat (and no other change or additional info)?
What kind of inhibitor is EDTA? Is it possible to recognize this mode of action using a Lineweaver-Burke graph? If yes, how?
EDTA is a chelator that traps divalent metal cations and thereby inhibits enzymes that need such ions for function. Mechanism not visible on Lineweaver-Burke. Will look like mixed inhibiton
Write down substrates and products of an ADH reaction
Ethanol + NAD+ --ADH--> Acetaldehyde + NADH + H+
You are performing an affinity chromatography purification like in laboratory 4. After collecting all the fractions, you take small aliquots of each to run them by SDS PAGE and check if you purified the protein efficiently by Coomassie staining. Explain shortly what might have happened in below scenarios Soluble lysate (after centrifugation to remove insoluble material) and flow through contain many protein bands (without a thick band in the size range of your protein of interest), there are a couple of thin bands in wash step 1, all further fractions contain no bands.
Even though there are a couple thin bands in the wash step, there is no band in other fractions, which means no POI is present in all these fractions. Therefore, POIs might not be expressed, or they are expressed but not in the fractions that mentioned above, which is the insoluble part. They aggregate and become insoluble. How could you discriminate between those two possibilities?
Describe the difference between first, second, and third generation biofuels
First generation: edible feedstock (corn) as energy source for biofuel production. - cons: competition with food production = impacting prices Second generation: plant waste material as energy source for biofuel production - cons: biomass in walls makes it harder to digest Third generation: algal cells engineered to produce biofuel by directly capturing atmospheric CO2 - pros: can be grown on land that is useless for agriculture, easy to grow, doesn't compete with feedstock
Why isn't the standard curve of Abs595 vs. protein concentration linear over a long range?
For Bradford specifically, the curve is not linear over a wide range because there is a decrease in the number of free red and green CBB and hence the background at higher protein concentrations. Here you should also think about what it is that you actually measure and if there might be some limitations that may lead to the curve flattening
Your roommate suggests you repeat the purification of alcohol dehydrogenase by affinity chromatography with ten histidine residues in the tag. Explain why this would or would not work.
I would argue that such a tag would still work but may be harder to elute the POI from the column due to the tight bond. The ten histidine tag would have a relatively higher affinity to the ligands in the column than other proteins and therefore can outcompete those during the washing step.
What could be alternative ways to determine the concentration of a single protein?
Immunoblot or ELISA (for antibodies/other POIs) or run enzyme assay (if POI is enzyme) or nanodrop (if protein is pure/wavelength=280 nm)
What is the difference between Ki and Ki'?
KI is the dissociation constant of inhibitor binding to free enzyme KI' is dissociation constant of inhibitor binding to enzyme substrate complex
What do the Km and Vmax indicate about an enzyme reaction?
Km indicates the affinity of the enzyme to a particular substrate Vmax indicates the maximum speed at which the enzyme can catalyze the reaction
For which purpose did you use the Bradford assay in the lab course?
Lab 5: Determine the concentration of purified YqhD
What are the different phases to a microbial growth curve, and what would it look like as a curve?
Lag phase: cells adapt to new environmental conditions by upregulating production of enzymes and proteins necessary for growth and division Log phase: cells are growing and dividing fast and contain high number of ribosomes Stationary phase: cell density gets so high that cells run out of space and nutrition and will turn off metabolism
Which graph would you pick to illustrate what 1st and 0th order reaction means? Label axes.
Michaelis-Menten graph where you can see 1st order reaction rates at limiting substrate concentrations and 0 order kinetics at unlimiting substrate concentrations
AFFINITY CHROMATOGRAPHY: . Remember the purpose of using relevant chemicals for this method (those that we covered in lecture). Which results would you expect if the incorrect molecule was used instead.
NPI-10,20,500: Bugbuster bacterial lysing reagent: Ni-NTA spin column
Do all proteins bind equally well to Coomassie Brilliant Blue dye?
No, mainly aromatic and basic side chains bind to CBB with higher affinity. All proteins bind but not with same affinity.
Can a primary and its secondary be from the same species?
No. Secondary antibodies are made when a primary antibody is injected into an animal. If it is from the same animal species, antibody molecules will not be considered "foreign" and won't illicit an immune response
Should you use Bradford assay at pH 7?
No. there won't be a color chance since CBB is already in blue form
Is it possible to use SDS PAGE to determine if a protein is found as a monomer, dimer, trimer, etc in the cell? Explain your answer.
Not if the monomers are connected via non-covalent bonds only. Treating proteins with SDS and reducing agent causes only the monomeric size to be seen on the gel. If the monomers are connected by disulfide bridges, you could see a difference when treating samples with or without reducing agent (B-ME or DTT). The protein in the samples not treated with reducing agent would run at the multimeric molecular weight, where proteins treated with reducing agent would run at the molecular weight range of the monomers
Draw a graph to illustrate what "initial rates" means. Label the axes. What is the issue with assays that are not in the initial rate?
Product formation (y-axis) vs. Time (x-axis) Initial rate is when only forward reaction is happening (linear portion of the graph)
Which components do you need on the plasmid for protein expression to work? List all components with their respective function
Promoter: to initiate transcription of gene of interest by RNA polymerase (which binds tightly to a promoter region) Origin of replication: binding site for DNA polymerase to initiate replication and hence to make copies of the plasmids that can be passed on to next generation Operator: binding site for repressor, which will block RNA polymerase from binding to the promoter and hence represses transcription of gene of interest (until we treat cells with induce to "de-press" transcription) lacI gene: encodes for the repressor protein, which binds to the operator and represses transcription of gene of interest until de-pressed Ribosome binding sequence: included on DNA to generate a ribosome binding site on mRNA, so that transcript can be bound by ribosomes for translation Selectable Marker: coding sequence for an enzyme (B-lactamase for ampicillin resistance). This enzyme cleaves B-lactam ring in ampicillin and detoxifies the antibiotic. Cells with this marker will survive antibiotic treatment, while cells without it will die. His6 tag: DNA sequence encoding for 6 histidine residues to be added to POI, which allows the POI to be purified by affinity chromatography Gene of interest: encodes for POI
Under which circumstances can you claim that the protein concentration measured in Bradford is of a single protein
Run SDS PAGE and check with Coomassie staining if only our target protein shows up. If there are multiple bands, then the protein concentration measured is probably not from the same protein
SDS PAGE/IMMUNOBLOTING: Write down all experimental steps and understand the purpose of each step. Which result for this experiment would you expect, if one of the steps was omitted (one by one)?
SDS PAGE: 1. remove cell pellets from freezer and thaw 2. dilute sample buffer from 5x to 1x 3. add diluted buffer to cell pellets - need to make time 1 and 2 normalized to have same amount of cell material in time 0, to load same number of cells into each lane 4. boil samples in heat block 5. Get OD to calculate how much volume 6. assemble gels and electrode assembly 7. fill inner chamber with running buffer 8. remove combs and then fill outer chamber with running buffer 9. load samples flanked by unstained and prestained protein molecular weight standard/ladder 10. run at 100 volts until samples reach separating gel, then run at 180 volts until dye front runs to ends (1 hour) 11. disassemble apparatus 12. cut gel, and place upper portion (above 50 kda) in Coomassie Blue staining solution. - this is loading control, will compare if you loaded samples evenly 13. microwave for 30 seconds, then incubate at RT with rocking for 5 min 14. pour off stain 15. rinse stained gel with destain soluition repeatedly until empty parts of gel are destained 16. rinse gel in water and you are done IMMUNOBLOTING: 1. same as above but when you cut gel, cut lower part (below 50 kda) for immunoblotting. 2. place gel in transfer buffer 3. make sandwich (nylon pad, two filter papers wet with transfer buffer, the gel, membrane, two filter papers, and nylon pad) 4. gel sandwich cassette goes into electrode assembly 5. frozen ice pack is added and tank is filled with transfer buffer 4. tank is in another container with ice then 100 volts for 1 hour 5. store membrane in TTBS - unbound primary antibody molecules will be washed away 6. membrane incubated in blocking buffer 7. TTBS is added and primary antibody is added (mouse anti-his6 IgG) 8. incubate for 1 hour 9. Add TTBS, wash with TTBS, and then add secondary antibody (goat anti-mouse IgG alkaline phosphatase) + TTBS 10. Incubate for 30 min. then wash with TTBS. 11. Add alkaline phosphatase buffer, incubate 12. Add fresh alkaline phosphatase buffer + NBT + X-P 13. Incubate in dark then you are done
How do you treat the proteins before loading them onto the polyacrylamide gel? Why?
SDS denatures and coats proteins with negative charges BME reduces disulfide bridges to further denature proteins Boiling accelerates denaturation
Why can we ignore the various protein conformations when comparing their migration in SDS PAGE?
SDS treatment disrupts non-covalent interactions stabilizing 3D conformation and reducing agents also reduce disulfide bridges
Why do all proteins migrate in the same direction in SDS PAGE, irrespective of their intrinsic charge?
SDS treatment evenly covers proteins in negative charge
You are performing an affinity chromatography purification like in laboratory 4. After collecting all the fractions, you take small aliquots of each to run them by SDS PAGE and check if you purified the protein efficiently by Coomassie staining. Explain shortly what might have happened in below scenarios Soluble lysate (after centrifugation to remove insoluble material) and flow through contain many protein bands (including a thick band in the size range of your protein of interest), there are a couple of thin bands in wash step 1, all further fractions contain no bands
Soluble lysate (after centrifugation to remove insoluble material) and flow through contain many protein bands (including a thick band in the size range of your protein of interest), there are a couple of thin bands in wash step 1, all further fractions contain no bands
What is the function of the T7 promoter on the plasmid DNA?
T7 promoter region is the recognition site for the T7 RNA polymerase for transcription of the gene of interest
Why don't we use Beer's law to analyze the Bradford assay?
We do not have molar absorptivity constant
When you plot the Michaelis-Menten graph, are all data points initial rates?
Yes
Are proteins able to bind to the Coomassie Brilliant Blue dye at pH 7?
Yes, proteins are still able to bind
What could be the issue if we used a first order enzyme assay for measuring enzyme concentration?
You would underestimate your enzyme concentration, as you limit the activity of the enzyme by limiting its substrate.
PROTEIN EXPRESSION: Remember the purpose of using relevant chemicals for this method (those that we covered in lecture). Which results would you expect if the incorrect molecule was used instead?
ampicillin: IPTG:
What part of a protein binds to the Coomassie Brilliant Blue dye?
binds to arginine side chains with high affinity. CBB also (basic and aromatic side chains) hydrophobic pockets will bind to CBB through hydrophobic effects
How is a mutation in the Lon protease helpful for protein overexpression?
by mutating Lon protease, all proteins are stabilized as they are protected by degradation from the protease
What are the options of visualizing proteins following separation by SDS-PAGE?
colorimetric detection: antibody conjugated to enzyme which produces colorful product fluorescence: antibody conjugated to a fluorescent molecule that can be excited and will emit light of a different wavelength that can be quantified chemiluminescent detection: enzyme conjugated to secondary antibody will catalyze reaction that results in light emission that can be quantified
Why is it useful to know the dissociation constant of the affinity pair utilized for affinity chromatography?
dissociation constant tells us how strong the interaction between the ligands and the protein is therefore, would influence the washing and elution step that follows if constant is too low, interaction is too strong, it would be hard to elute proteins off column if constant is too high, interaction is not strong enough, it would be hard to separate our POI from other proteins
How can you quantify protein concentrations in Bradford assay?
generate Standard Curve using known concentrations of proteins, measure absorbance at 595 nm of unknown sample and plot it on the curve to find out its concentration
You are working in a biochemistry lab and are analyzing an enzyme. If you know the molarity of the enzyme, would you be able to determine its specific enzyme activity or its turnover number? Describe the difference between the two
if you know the molarity of the enzyme, you can test how fast the enzyme works in producing product per mol enzyme. while the specific enzyme activity describes how fast the enzyme produces product per mg enzyme
What happens if you induce a culture in the lag phase?
in lag phase, cells are sensing the change in environment (more nutrients and space) so they are preparing to enter exponential growth phase by transcribing and translating the necessary proteins/enzymes for cell division and growth. forcing cells to produce foreign proteins when they are not yet growing fast will use a lot of the cells' resources for their own proteins to produce the POI, therefore inhibiting cell growth and will either delay or prevent cells from reaching log phase. since you are relying on cells multiplying to produce more protein expression, it is important that cells reach log phase and divide quickly.
What is the function of the operator sequence on the plasmid DNA?
it is a regulatory site for transcription and the repressor can regulate the transcription of gene of interest by binding or leaving such a region (binding -> preventing the RNA polymerase from transcribing the gene of interest). (IPTG binds repressor, which changes conformation -> does not bind the operator, allowing for transcription) allows for transcription of gene when IPTG is added to be induced
While we discussed the advantage of using longer chain alcohol molecules for biofuels, what could be one reason for industry to still produce large amounts of ethanol as biofuel?
it is still so much easier technologically to produce ethanol by simply depriving cells of oxygen
What does inducing protein expression really mean? And how does inducer contribute to the control of the expression of the gene of interest?
it really means releasing the repression of the transcription by removing the repressor that is bound to operator close in proximity to promoter. when the inducer IPTG is added, it will bind to repressor, change conformation, and no longer bind tightly to operator
What happens if you induce a culture in the stationary phase?
metabolism switches off due to reduced space and decreasing food supply then expression of POI is less efficient than during log phase
What is the nature of the interaction between the ligand and target in an affinity pair?
noncovalent bond accumulation which provides a strong enough interaction between the affinity pair. others include: electrostatic, hydrogen bond, hydrophobic interaction, complementary shape
Explain what is different in the separating gel (compared to the stacking gel) that allows proteins of different sizes to migrate at different speeds.
pH: higher pH (8.8) to make glycine negatively charged again to stop stacking effect Acrylamide/bisacrylamide Concentration: higher concentration to decrease pore size for separation of proteins by molecular weight
What are the two types of ladders you used for immunoblot and SDS PAGE? Why did we use one over another for each respective technique
pre-stained ladder: used this for immunoblot since the reference bands must be stained with something and the antibodies we used are only going to bind to specific proteins (namely YqhD) unstained ladder: proteins are not conjugated to dye molecules, but since all proteins are stained by Coomassie Brilliant Blue, also ladder proteins will be visible. unstained proteins run more uniformly on the gel and will form more precise bands.
What is the difference between primary and secondary antibody molecules?
primary antibodies bind to target protein specifically (via variable region) secondary antibodies bind to constant region of the primary antibody (that is their antigen)
What is cell density and how do you measure it?
refers to number/amount of cells in a given unit of volume. measure it by measuring absorbance at 600nm. OD600= 1 is 10^9 cells per mL
What is the purpose of running SDS-PAGE?
separating proteins by molecular weight - useful when analyzing composition of protein extract or assessing purity of protein in solution (when stained in Coomassie)
What would happen if the pores in the stacking gel were as small as those in the separating gel? What would the gel look like?
smaller proteins would pass more quickly through the stacking gel and stacking effect would not get proteins of all sizes to the start of the separating gel at the same time. essentially, stacking effect would fail
Describe the rationale of how immunoblotting works
specific antibody only binds to target protein location of antibody-target complex is visualized by using a secondary antibody binding specifically to constant region of primary. secondary is conjugated to an enzyme or fluorescent dye, so that the location of the target protein can be seen either by a colorful precipitate, light emitted from a chemical reaction, or a fluorescence that is emitted after the dye molecule is excited
Explain why we need a stacking gel and how the stacking effect works.
stacking gel s are used to compress the proteins in the sample to a thin line before they enter the separating gel. otherwise, not all proteins of the same molecular weight will reach the same location in the gel and won't form a focused band Stacking effect relies on glycine zwitterions that create a voltage differential across the protein field by increasing the local voltage "above" the proteins (trailing ions). This high local voltage results from a high resistance in the area of the gel with a high amount of relatively immobile glycine zwitterions (Ohm´s law). It is important that the glycine anions are protonated (now zwitterions) when they enter the stacking gel, acting as trailing ions. They will only exhibit the high resistance (->high voltage) in their zwitterionic form. In order to enter the gel, they will have to be negatively charged in the buffer (therefore the pH there has to be more basic). Chloride ions act as leading ions. They are already part of the gel and will migrate to the positive electrode once the electric field is turned on. Their high mobility will lead to a low resistance in that part of the gel ->low local voltage in this part of the gel. Proteins that are higher up in the well will be closer to the high local voltage by the glycine zwitterions and will migrate faster in the stacking gel than the proteins closer to the Cl- ions (further down compared to other proteins). These proteins are slower than the ones above, because they are closer to the low local voltage due to the low resistance from the Cl- ions. This way the proteins that ended up on the top of the well will migrate faster than those on the bottom of the well and catch up by the time they reach the separating gel. Entering the separating gel, glycine ions will be deprotonated due to a higher pH and run ahead of the proteins due to their negative charge. Proteins are not pushed any more and can separate by their molecular weight.
How do you adjust the pore size in an acrylamide gel?
the higher the ratio of acrylamide to bisacrylamide = the higher the total concentration = the more crowded the gel = smaller the pores
How do you transfer the experimental data of product vs. time to the Michaelis-Menten graph?
the linear part of the slope in the product/time graph is plotted for each substrate concentration in the Michaelis-Menten graph
Why do you need to monitor the cell density of various samples?
to be able to normalize samples with each other and load equal cell number or total protein amount per well
What is the purpose of affinity chromatography? Keep in mind the alternative methods (discussed during Week 8/9).
to select the POI through the difference in binding affinity POI should have significantly higher affinity to ligands than other proteins, such that we can wash off other proteins and retain our POI binding to the column and elute them afterwards. Immobilized Ni+2 in matrix binds to the His6 tag attached to POI and thereby catching the POI in the column while all other proteins will be washed through faster
Why do you need a loading control?
to show that all samples were normalized correctly and loaded evenly so that samples can be compared for abundance of POI fairly
How can modern biotechnology be applied to produce biofuel molecules?
we can use microbes as factories that produce various biofuel molecules when overexpressing the respective enzymatic pathways
Which elution method did you use in the lab? How/why did this work?
with a higher concentration of imidazole which could out compete the binding of nickel with the POI, allowing the POI to elute out from the column addition of competitor with target molecule - imidazole in histidine tag
Specific enzyme activity. You are measuring the maximum velocity of a dehydrogenase enzyme by looking at the absorbance of 340 nm. During the initial rate phase, you measure an absorbance change of negative 0.5/min. The molar absorptivity coefficient is 6.22 mM-1 cm-1 and the cuvette length is 1 cm. The kcat of this enzyme is 1300 min -1 . The original undiluted enzyme stock concentration is unknown. You dilute the enzyme by the dilution factor of 15 before adding it to the assay. You then used 18 µL of this diluted enzyme solution for the enzyme reaction in a total volume of 1.3 mL. What is the original molarity of the enzyme?
ΔA = ε • l • ΔC 0.5 min -1 = (6.22 mM-1 •cm-1 )(1 cm)(ΔC) ΔC = 0.08 mM Product/min Given kcat = Vmax / [ET] = 1300 min -1 [ET] = (ΔC) / kcat (0.08 mM Product/min) / (1300 mM Product/mM enzyme • min) [ET] = 6.18 x 10 -5 mM In cuvette/assay: (make sure to bring both volumes to same unit - either to µL or mL) M1V1 = C2V2 ⇒ (1300 µL)(6.18 x 10 -5 mM) = (C2 )(18 µL) ⇒ C = 4.5 x 10 -3 mM Accounting for 25X dilution: C = 4.5 x 10 -3 mM • 15 = 6.7 x 10 -2 mM ∴ The original molarity of the enzyme is 6.7 x 10 -2 mM.