BIO 3C Lab Quiz 2

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Use the standard curve equation generated to calculate the molecular weight of the bands seen in the sample lanes. * Migration distance is on the x-axis, log of MW (kDa) is on the y-axis. -0.327x + 2.46 With the molecular weights of the samples calculated, determine the approximate number of amino acids that ech protein contains.

* 110 daltons per amino acid.

Isolation of Plasmid DNA by Mini Prep Calculate the concentration of DNA isolated (given the absorbance values). Calculate the total amount of DNA isolated (the "yield") for each miniprep. * You combined 2 µL DNA and 98 µL Buffer EB.

* 2 + 98 = 100 -> 2:100 -> 1:50 dilution -> 50 is the DF.

Tyrosinase Enzyme Kinetics Dopachrome absorbance at different enzyme concentrations.

* Amount of dopachrome formed was discovered by using the standard curve mentioned prior after measuring absorbance. * A single substrate concentration was used. * Higher absorbance reading means more dopachrome was formed.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) IP Sample: Would it have been possible to isolate GFP from DH5α E. coli cells that had not undergone transformation? Why or why not. Select a protein that could have been isolated by immunoprecipitation without prior transformation and cite your information resource.

* E. coli doesn't make GFP by itself.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) 1. Provide a separate photo of the Coomassie-stained gel with the lanes labeled and the migration distances (in cm) for all lanes (crop out ruler for this figure). Distances for the standard should be on one side, while the distances of the protein bands should be on the other. Numbers should not be written on the picture itself. 2. Provide a photo of the Coomassie-stained gel with the lanes labeled and the band sizes (in kDa) for all lanes. Sizes for the standard should be on one side, while the sizes of the protein bands should be on the other. Numbers should not be written on the picture itself. Use the kDa sizes that are already known (provided in Table 3 below).

* Expect one band for each sample, except for the antibody sample, which should have two bands (heavy chain and light chain). - 2 heavy chains migrate together and 2 light chains migrate together (even though there is four chains, there looks like two).

Bacterial Transformation What is the purpose of the control reaction where no DNA is added?

* If the cells are growing on the selective media without plasmid, maybe the cells already have ampicillin, or the ampicillin isn't working correctly.

DNA Electrophoresis What technique should you use to fill the wells?

* Leave lanes 1 and 8 empty. Insert the pipet tip just into the well; be careful not to poke through the thin agarose layer at the bottom of the well. Go to the first stop to eject, remove the tip from the buffer before slowly releasing your thumb. Slowly pipet the sample into the well and watch to see that it settles in the bottom of the well and does not spill over to other wells or on top of the gel. * Aim for the center. Go left -> right to feel resistance and then push liquid in. 1st stop then 2nd stop (if needed), pull the tip out.

Immunoprecipitation What does an antibody look like? Be able to label and draw one. * What is the antigen-binding region of the antibody?

* The Fab region is the antigen-binding region of the antibody.

DNA Electrophoresis How much agarose and 1X TAE buffer would be needed to prepare 40 mL of agarose gel at the following concentrations? 1. 0.8% 2. 1.2% 3. 3.0%

* We don't adjust the volume of buffer based on the powder for agarose gels, for all of these questions the volume of buffer needed is 40 mL.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Use the migration distance measured along with the known molecular weights of the protein standards to make a standard curve (see Figure 4). Graph migration the log of MW versus migration distance. Add a best-fit line and display the equation and r-squared value on the chart.

* the heaviest move the slowest.

DNA Restriction Digest Provide a labeled picture of your agarose gel under UV illumination.

* uncut plasmid: "uncut supercoiled" migrates faster than digested DNA and "nicked" migrates more slowly.

DNA Electrophoresis If you have 30 mL of 1X TAE buffer and you want to make a 0.7% agarose gel, how would you calculate the # grams needed to make this gel?

* x% = xg/100 mL

Tyrosinase Enzyme Kinetics *** Random Rules ***

*** DO NOT USE ABS VALUES >1 *** *** THE BLANK DOES NOT RECEIVE ANY SUBSTRATE *** *** ONCE ENZYME IS ADDED TO SUBSTRATE, IMMEDIATELY INSERT INTO SPEC (time = 0) *** *** REBLANK EACH TIME IF OTHERS HAVE USED THE SPEC *** *** IF YOU HAVE TO MAKE A NEW ENZYME DILUTION, MAKE A NEW BLANK AS WELL (blank contains enzyme dilution) ***

Tyrosinase Enzyme Kinetics Based on your estimate of the KM for tyrosinase and compared to other known enzymes, does tyrosinase require a large or small concentration of substrate to yield product?

*** Remember that a low KM value indicates the reaction is at ½ of Vmax at a low concentration of substrate. *** My Km value was way bigger than the Km for the other enzymes. This means that tyrosinase has a lower affinity for its substrate and requires a larger concentration of substrate to yield product, and achieve Vmax.

DNA Electrophoresis How would you go about calculating how much loading dye to add to each tube when the initial loading dye concentration is ___ (not sure it will be given, look at the answer and memorize initial loading dye concentration)?

*** Typical final concentration of loading dye is 1X, loading dye initial concentration was 6X). *** - We knew to use 12 microliters as the final volume because we were using multiples of 6 since the initial concentration is 6X. We didn't use 6 microliters because we know that the final volume has to be greater than 10 microliters since the volume without the loading dye is already 10 microliters.

DNA Restriction Digest What are the expected fragment sizes for the following pair of enzymes used to cut pGLO. Use the plasmid map given in the lab. * BstEII and Xhol * PstI * BamHI and ScaI

*** WATCH FOR ENZYMES THAT CUT MORE THAN ONCE ***

Isolation of Plasmid DNA by Mini Prep Uses of DNA cloning

-Biopharmaceuticals. -Gene therapy. -Gene analysis.

DNA Electrophoresis Calculate the fragment mass of the 2322 bp fragment in the λ HindIII digest lanes (3-5). Show your calculation. * Lane 3: total micrograms DNA was 0.5 * Lane 4: total micrograms DNA was 1.0 * Lane 5: total micrograms DNA was 2.0

.

Immunoprecipitation Draw a workflow for today's lab.

1. Add 10 microliters anti-GFP antibody (2 micrograms) into the lysate. 2. Place on rotator in cold room and rotate and incubate for 45 min. * Antibody is connecting to protein of interest. 3. Obtain IP tube and add 20 microliters Protein A/G beads. 4. Place on rotator in cold room and rotate and incubate for 30 min. * Beads will connect to antibody/protein-of-interest complex. 5. Spin down 1000 x g (3500 rpm) for 4 min in cold room. * Complex settles at bottom. 6. Remove supernatant and discard. 7. Add 1 mL 1xPBS and spin again 4 minutes, 3500 rpm. * Washing 8. Repeat steps 6 and 7 above for 2 additional spins (3 washes with PBS total). 9. Remove PBS carefully and leave minimal layer of PBS above the beads (around 10 microliters). 10. Add 25 microliters 2X FSB. * This denatures and contains dye for tracking. 11. Boil at 100 degrees Celsius for 5 min. * This further ensures separation by denaturing. * Antibodies are released from the beads. 12. Spin 1 min at top speed in the centrifuge. * Beads settle at the bottom, protein and antibodies are in the supernatant. * Antibody remains in supernatant, but it's okay because we will see the heavy and light chains of the antibody AND GFP in SDS (we will be able to see that only the protein of interest and antibodies are left). 13. Remove from heat block and bring to front. 14. Supernatant gets analyzed by SDS.

Isolation of Plasmid DNA by Mini Prep What is one of the most common methods used to isolate plasmid DNA from bacteria?

1. Alkaline lysis of bacteria 2. PH neutralization 3. Binding of DNA to silica membrane 4. Alcohol wash 5. Elution of DNA from silica membrane

DNA Electrophoresis In order to keep track of which tube has already received a particular reagent, the trick is to _________ the tube to a new _______ in the eppi rack.

1. move 2. location

Isolation of Plasmid DNA by Mini Prep Tell me where the plasmid DNA is in the following steps: 1. After pipetting 1 mL of culture into each tube and centrifuging.. 2. After adding N3. 3. After transferring the supernatant to a QIAprep spin collumn, and centrifuging. 4. After adding EB buffer.

1. pellet 2. supernatant 3. silica membrane in the blue column. 4. in the buffer EB

DNA Electrophoresis * What is it? * How does it work?

A method used for the separation and analysis of molecules. It uses the principle that charged molecules will migrate in an electric field. The primary factors which influence a molecule's migration rate are: number of charged groups on a molecule, molecular weight, shape of the molecule, and viscosity of the medium used for separation. In this exercise, we used the technique of agarose gel electrophoresis to separate DNA fragments of various sizes. To separate the DNA fragments, the samples are loaded on a stabilizing medium (agarose) which serves as a matrix for the buffer in which the molecules travel. The agarose gel, containing pre-formed sample wells, is submerged in buffer that is contained within the electrophoresis gel box. Samples to be separated are loaded into the sample wells. Current from the power supply travels to the negative electrode (the cathode) supplying electrons to the conductive buffer solution, gel, and positive electrode (the anode), thus completing the circuit. Electrophoresis buffer systems are designed so that the biological molecules of interest (DNA) have an overall negative charge. For instance, at neutral pH, a molecule of DNA is negatively charged due to the negative charges on the phosphate backbone. Under these conditions, samples applied to the sample wells at the negative electrode end of the gel migrate as zones within pores of the gel matrix towards the positive electrode. The agarose gel serves as a molecular sieve in that its structure is similar to that of a sponge. During the electrophoretic run, large molecules move more slowly through the gel than small molecules. Thus, the method sorts the molecules according to sizes since it relies on the ability of uniformly charged molecules to fit through the pores of the agarose gel matrix.

Tyrosinase Enzyme Kinetics In Part 3, Step 10, why is a new blank prepared for the second test sample?

A new blank was prepared for a second test sample because a new blank must be prepared when testing a different enzyme dilution. Once we decided on our 1:1 dilution to use for the rest of the lab, we were able to reuse the same blank. * Remember, the blank includes the enzyme dilution, so if you change the enzyme dilution, you must also change the blank so that it includes the new enzyme dilution.

Isolation of Plasmid DNA by Mini Prep Background info * Satellite colonies

A plasmid preparation always begins with the growth of the bacterial culture, lysis of the bacteria, and ends with purification of plasmid DNA (while discarding genomic DNA). Plating a transformation reaction (as performed in the previous lab) on selective media, such as LB-AMP, ensures that only the bacteria which contain the plasmid will grow. The plasmid is the only source of ampicillin-resistance genes which allow the bacteria to grow in the presence of the antibiotic ampicillin (because only the stuff containing the plasmid are resistant to ampicillin and thus, able to survive). Typically other genes of interest are also included on the plasmid, and after transformation a few colonies are selected to grow in larger cultures for the purpose of isolating the plasmid DNA. In this way, bacteria amplify plasmid DNA and the encoded genes of interest. The initial colonies that grow express ampicillin resistance genes and inactivate the ampicillin in their immediate vicinity. If the cultures overgrow, bacteria that lack the plasmid may be able to grow beside bacteria containing the plasmid, relying on the transformed bacteria to inactivate the ampicillin. These non-transformed bacteria often form smaller colonies that are very close to the transformed bacteria, and are referred to as satellite colonies. Satellite colonies often form a halo around the larger transformed colonies. It is important to avoid satellite colonies since they do not contain the plasmid of interest. If a culture is inoculated with both transformed and non-transformed bacteria the non-transformed bacteria may have higher fitness and take over the culture since they do not have to replicate and express plasmid DNA. Such cultures would result in poor yields of plasmid DNA. After bacteria are transformed and grown on selective media, a few colonies are selected for mini-culture and further analysis to see if they contain the plasmid introduced in transformation. A mini-culture is typically 3 mL, and provides enough DNA to analyze by restriction enzyme digest and/ or DNA sequencing. When setting up bacterial cultures it is important to use aseptic technique so that contamination from the air and bench top is not introduced; nor is the work space contaminated with bacteria.

DNA Electrophoresis How is DNA visualized after electrophoresis is performed? * How does this work?

After electrophoresis is performed, the DNA is visualized by adding an intercalating dye such as ethidium bromide. In this lab we will use Sybr Safe DNA stain (BioRad), which is an alternative to ethidium bromide and is not carcinogenic. These dyes insert between the DNA double helix and fluoresce green when illuminated by UV light (260-360 nm). A record of the agarose gel can be made by taking a picture using a camera.

What type of gel do we use to separate DNA? What about separating proteins? What are other differences between these gels?

Agarose -> separate DNA SDS gel -> separate proteins * Agarose gel used 1 kb ladder. * SDS gel uses protein standard ruler, which is prestained. The protein standard uses known sizes of different proteins.

Isolation of Plasmid DNA by Mini Prep What is another term for DNA Mini-Prep?

Alkaline lysis

Bacterial Transformation Waste disposal

All materials that touch live culture (E. coli cells) must be disposed of as biohazard. Any eppi tubes and tips used for bacteria or the transformation reaction should also be disposed of in the biohazard waste bucket.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Why didn't you prepare a tube #1? Why did you load directly from the stock tube into the lane?

Because the Protein Standard that goes into lane 1 is already prepared. You don't need to add FSB to it, nor boil it like the other tubes.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) * Know how to do a calculation to prepare your sample for loading onto an SDS-PAGE gel. EX: You need to load 5 micrograms of your ovalbumin sample. The concentration of your stock volume is 0.89 micrograms/microliter. * Do you need to dilute your sample? Or can you load straight from the stock you have? You sample volume must be at least 5 microliters.

Before loading a sample onto an SDS-PAGE gel, it must be diluted to an appropriate concentration (i.e. 0.1-10 mg/mL) in electrophoresis sample buffer containing several key ingredients. * If you need to make a dilution, use C1V1 = C2V2 to calculate the volume needed for a final concentration of 1 µg/µL. Make 100 µL total. If your V1 is less than 1 µL, repeat the calculation so that your V2 is 200 or 300 µL. MY EXPERIENCE: 0.89 µg/µL x _________= 5 µg. * 5.62 µL needed.(5/0.89 = 5.62) - I don't need to dilute since I have more than 5 µL. I would only need to dilute if I had less than 5 µL, because we're adding 5 µL + 5 µL FSB to get 10 µL total (minimum). Since I have more than 5 µL (5.62), my total volume is adjusted to 10.62. I would only need to dilute if I don't have enough volume. DILUTION EXAMPLE * Example of someone that needs to dilute. 20 µg/µL x ______ = 5 µg. - 0.25 µL needed (too low). C1V1 = C2V2 (20 µg/µL)(V1) = (1 µg/µL)(100 µL) V1 = 5 µL * 100 - 5 = 95 µL sterile water. * Mix these together to make the dilution, and take 5 µL out of the 100 µL dilution mixture.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) How do you prepare a sample for SDS-PAGE?

Before loading a sample onto an SDS-PAGE gel, it must be diluted to an appropriate concentration (i.e. 0.1-10 mg/mL) in electrophoresis sample buffer containing several key ingredients. Final Sample Buffer (FSB) Components: 1. SDS - denatures protein and provides an overall negative charge 2. β-mercaptoethanol - reduces disulfide bonds 3. Tris-HCl - adjusts the buffer to the same pH and concentration as the electrophoresis running buffer 4. Glycerol - adds density to the sample and causes it to sink to the bottom of the sample well 5. Bromophenol blue - a "tracking dye" that runs faster than most proteins, allowing the progress of the electrophoresis to be visually monitored Usually samples are heated in a heat block for 5 minutes prior to loading on the gel. Heating the proteins in the presence of SDS causes the proteins to denature, while the β-mercaptoethanol reduces the disulfide bonds, further ensuring protein denaturation.

Isolation of Plasmid DNA by Mini Prep EB

Buffer EB: DNA is commonly stored in EB buffer ("elution buffer"), which buffers against pH change that could damage and denature the DNA. It contains 10mM Tris-Cl at pH 8.5. Our kit names this solution "EB."

Tyrosinase Enzyme Kinetics Know and understand how to make dilutions.

C1V1 =C2V2 (1/1)(V1) = (Desired dilution. ex: (1/10))(5 mL)

Isolation of Plasmid DNA by Mini Prep Summarize the steps of DNA cloning:

Cutting and pasting DNA using two enzymes: restriction enzymes and DNA ligase. Bacterial transformation and selection. The plasmid contains an antibiotic resistance gene which allows bacteria to survive in the presence of a specific antibiotic. The antibiotic we used in Lab #8 was ampicillan. Protein production. Bacteria serveas factories to produce large amounts of protein, but the protein must be purified.

Isolation of Plasmid DNA by Mini Prep Define: *DNA cloning *Plasmid *Transformation

DNA cloning: a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene. Plasmid: In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid. Transformation: The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.

Bacterial Transformation Know the Central Dogma of Molecular Biology

DNA replication: the process by which the genome's DNA is copied in cells. Transcription: the process of making an RNA copy of a gene's DNA sequence. Translation: the process through which information encoded in messenger RNA (mRNA) directs the addition of amino acids during protein synthesis. * Replication: DNA -> more DNA (genomic DNA copied) * Transcription: DNA -> mRNA * Translation: mRNA -> proteins

DNA Electrophoresis How are DNA samples prepared for DNA electrophoresis? * Know the components and their functions.

DNA samples are prepared by mixing a small amount of DNA, usually around 0.1 μg, with a loading dye. The loading dye contains glycerol, xylene cyanol, bromophenol blue, & orange G. The glycerol makes the sample mixture denser than the running buffer, allowing the sample to sink to the bottom of the well once loaded in the gel. Two blue dyes are typically used, xylene cyanol & bromophenol blue, along with a yellowish dye orange G. These dyes allow visualization of the electrophoresis progress. Xylene cyanol migrates with larger DNA fragments, around 4 kb (4000bp); bromophenol blue migrates with smaller DNA fragments, around 0.3 kb (300 bp); and orange G migrates with DNA fragments around 50 bp in size.

Tyrosinase Enzyme Kinetics Let's say your absorbance reading isn't satisfying you, what should you do?

Depending on the absorbance readings, make a second enzyme dilution either more/less dilute. * Too slow -> more concentrated. * Too fast -> more dilute. * Dilute the enzyme extract with an appropriate volume of citrate buffer. For example, adding an equal volume of buffer (1:2 dilution) should approximately double the time of the reaction.

Bacterial Transformation What protein first binds to oriC locus?

DnaA

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Electrophoresis Background

Electrophoresis is the movement of charged ions and molecules in an electric field. Molecules with a net positive charge will migrate toward the negative electrode (cathode), while those of net negative charge will migrate toward the positive electrode (anode). The velocity of movement depends on several factors expressed in the equation below: v = q E / f where v = velocity q = net charge on the molecule E = applied voltage f = frictional coefficient Thus, movement of the molecules is dependent on the pH of the system (which affects the charge on various functional groups), the size and shape of the molecules, the support medium used for the separation, and the applied voltage. For example, if voltage is increased, the samples will separate faster; however, exceeding the appropriate voltage may damage the sample or the separation medium due to excessive heating. The frictional coefficient for molecules separated by electrophoresis is a function of the retarding effect of the support medium and the size and shape of the molecules. A round protein, for example, will move faster than a rod-shaped one with the same molecular weight. A denser medium retards all proteins, but has a greater effect on larger proteins. Many different separation media are employed for electrophoresis including liquid, paper, and gels. The most commonly used media for protein or nucleic acid separations are gel-based media. A protein mixture dissolved in a buffer solution is applied to the surface of the gel medium (i.e. agarose or polyacrylamide gel) in an electrophoresis apparatus. The gel is in contact with electrophoresis buffer that completes the electric circuit between the electrodes at each end of the electrophoresis cell. In many cases, the pH of the electrophoresis buffer is above the isoelectric point (pI) of the molecules in the mixture, thus ensuring that the molecules all have a net negative charge. When electric current is applied to the field, the proteins will migrate at variable rates through the gel matrix, creating zones or bands of proteins. The zones or bands can be made visible by staining the gel matrix with protein-binding dyes at the end of the electrophoretic run.

Immunoprecipitation How is the Fab portion of the antibody different from the Fc portion?

First off, the Fab portion is the antigen-binding region, while the Fc portion is not. Also, they are different domains. The Fc portion is only constant heavy chains while the Fab portion is a mixture of constant, variable, heavy and light chains.

DNA Electrophoresis What is the equation we use to calculate fragment mass?

Fragment mass = (fragment length bp x total amount DNA)/undigested DNA length)

Isolation of Plasmid DNA by Mini Prep P1

Glucose/Tris/EDTA (GTE): Glucose in this buffer maintains osmotic pressure, Tris provides a pH buffer around a pH of 8.0, and EDTA binds divalent cations in the cell membrane, thus weakening it. After the cells are denatured, EDTA chelates other metals such as Mg2+. Magnesium is a necessary cofactor for DNA nucleases found in bacteria, therefore by binding magnesium EDTA helps to prevent DNA degradation. Our kit names this solution "P1." * EDTA keeps bacteria separated, prevents sticking. It also helps DNAse from hurting DNA

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) If the SDS-PAGE analysis of the IP shows other proteins in addition to the protein of interest, what step of the IP protocol should be modified?

If the SDS-PAGE analysis of the IP shows other proteins in addition to the protein of interest, I think that there should be additional rinsing steps of the beads with the buffer to get rid of any unwanted proteins.

Bacterial Transformation Explain how arabinose affects the ability of the bacterial cells to produce GFP.

In the presence of arabinose, the AraC protein promotes the binding of RNA polymerase to the promoter, which causes transcription of the GFP gene into messenger RNA (mRNA), followed by the translation of this mRNA into GFP.

Bacterial Transformation What is the function of the pBAD promoter?

It binds AraC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene.

Bacterial Transformation What is the function of the araC gene?

It encodes the regulatory protein that binds to the pBAD promoter; only when arabinose binds to the AraC protein is the production of GFP switched on.

Tyrosinase Enzyme Kinetics Why is it important to add enzyme right after the spec has been zeroed out?

It is important to add the enzyme right after the spec has been zeroed out because our goal is to get the solution in the spec immediately, at time zero. If we didn't prepare the spec before putting the enzyme in, the enzyme would be reacting with the substrate outside of the spec, which is not good.

DNA Restriction Digest Why did the uncut DNA in the fourth lane at 5,371 bp appear smaller in size than the 3695 bp fragment from the digestion reaction?

It's circular, so it is more compact. It is supercoiled.

Bacterial Transformation What is a promoter?

It's where RNA polymerase binds the DNA and begins transcription of the gene.

Isolation of Plasmid DNA by Mini Prep Bacteria with the correct plasmid are used to:

Make more plasmid DNA (which is what we did in lab #9). Express the gene to make protein (which is commonly done in labs in order to understand protein function). In lab #8, we added pGLO plasmid to induce expression of GFP.

Tyrosinase Enzyme Kinetics Substrate Specificity of Tyrosinase

More product was formed when DOPA was the substrate. DOPA is preferred over pyrocatechol and hydroquinone due to its shape being more similar to physiological substrates of tyrosinase. The alternate substrates are still leading to the production of dopachrome.

DNA Electrophoresis Does the ruler need load dye?

No. It already has dye.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Polyacrylaminde-Specific Electrophoresis * What are the benefits of SDS-PAGE?

Polyacrylamide gels are formed by the polymerization of acrylamide cross-linked with N'-N'-methylene-bisacrylamide. Long strands of cross-linked polymers are formed resulting in a gel containing many pores. The size and number of pores are dependent on the concentration of acrylamide and the amount of crosslinker added. Most polyacrylamide gels are poured as a "slab" between two plastic plates. Most protein electrophoresis systems use gels made of two parts: an upper "stacking" gel and a lower "resolving" gel. The stacking gel is a few millimeters high and is made of a low percentage (e.g., 4%) acrylamide at pH 6.5. It is called the stacking gel because samples tend to compress into a thin band. The resolving gel, the majority of the gel, is a higher percentage acrylamide (7.5% - 15%) at pH 8.5. The resolving gel (also called the separating gel) is the portion of the gel where the proteins separate, with the smaller proteins running faster. The stacking gel works in two ways. First, it is a low-concentration gel, so that proteins move relatively quickly through it. When the proteins encounter the higher-density resolving gel, they slow down. The proteins that go into the resolving gel first are going slower than the proteins still in the stacking gel. This creates an accordion-effect, causing the protein sample to compress as it enters the stacking gel. Second, the pH difference between the stacking and resolving gels affects the overall charge on the sample molecules. The buffer system contains Tris-HCl and glycine. The glycine in the buffer at pH 6.5 is a zwitterion (both carboxyl and amino groups are charged) with an overall net 0 charge, so that it migrates relatively slowly. Within the resolving gel of pH 8.8, the overall charge on the glycine molecule becomes negative, thus relieving the stacking effect. At this point, the various proteins migrate according to their molecular weight, overall shape, and charge differences. Amino acids exist in different charge states, depending on the environment. At physiological pH amino acids are zwitterions (have a positive and negative charge). Glycine acts as a current-carrying anion in SDS-PAGE and is present in the buffer. Glycine from the buffer enters the sample buffer and the gel itself and becomes neutral in the stacking gel with a pH of 6.5 because its amino group is protonated (NH3+) and its carboxyl group (COO-) remains deprotonated. In the resolving gel (pH of 8.9) most of the glycine molecules take on a negative charge (amino group acts as proton donor: NH2) and migrate through the gel. * Glycine helps carry the current. SDS makes the proteins negative, but glycine helps carry the negative proteins to the positive end. A newer development in polyacrylamide gels is gradient gels, which we will use in our lab exercise. Gradient gels are cast using advanced machinery and provide a continuous gradient from the loading region to the bottom. A typical gradient is 4-20% and allows a wider range of protein sizes to be detected relative to a single percentage gel. Our gradient gels allow detection of proteins between 10-100 kDa. Since electrophoresis separates proteins as a function of their molecular weight, shape, and charge, it may be difficult to interpret results of native proteins. When we wish to determine the molecular weight of proteins, the detergent sodium dodecyl sulfate (SDS) is employed to provide the protein with an overall negative charge. Usually a small amount of β-mercaptoethanol is included in the sample buffer to ensure that disulfide bonds are broken. The effect of adding β-ME and SDS to the sample is that all proteins assume a denatured (random coil) conformation and have the same charge/mass ratio. Thus, the only variable left is mass. In SDS-PAGE, proteins separate in the gels solely based on molecular weight. SDS-PAGE is often used to determine if a protein is pure and if it is made up of subunits. For example, using gel filtration you might determine that the molecular weight of a protein is 100,000 daltons. If you ran the same protein on SDS-PAGE and observed bands and 30,000 and 50,000 daltons, you could conclude that the protein contains two, differently sized subunits.

Isolation of Plasmid DNA by Mini Prep N3

Potassium acetate/acetic acid (KOAc): Acetic acid neutralizes the pH following alkaline lysis of the bacteria. The DNA renatures, forming dsDNA; however the large chromosome becomes tangled during this process while covalently closed plasmid DNA readily reanneals, forming the correctly paired superhelical structure and remaining in solution. The potassium acetate precipitates SDS along with associated proteins and lipids. The precipitated proteins, lipids, & chromosomal DNA will be removed by centrifugation while the plasmid DNA remains in solution for further processing. Our kit calls this solution "N3."

Bacterial Transformation What is the purpose of the recovery step?

Recovery allows cells time to copy the transformed plasmid and express genes encoded by the plasmid before placing the bacteria on selective media. * Allows more cells to become transformed

DNA Restriction Digest Background

Restriction endonucleases, also called restriction enzymes, were discovered in the 1950's-1970's and are found in many bacterial species. The natural function of restriction enzymes is to protect bacteria from viral infection. Restriction enzymes recognize and cleave DNA, so when DNA is ejected into a host cell during the initial stages of viral infection, restriction enzymes recognize and cleave specific regions of the viral DNA, thus inactivating it. The cut DNA is said to be "restricted." There are three types of restriction endonucleases (I, II, & III). Type II endonucleases are the simplest, do not require ATP, and cleave DNA within a specific recognition sequence. Their recognition sequences are typically 4-6 base pairs (bp) long, and are palindromic, meaning that the sequence 5'-to-3' is the same on both strands. Type II endonucleases are among the most commonly used enzymes in molecular biology. In this lab we will work with 2 restriction endonucleases, EcoRI & Eco321. Note their recognition sequences and cut sites in Figure 1. EcoRI makes a staggered cut on the DNA sequence leaving a 3-4 nucleotide (nt) overhang. This overhang is referred to as a "sticky end." Other type II restriction enzymes cleave both strands at the opposing phosphodiester bond leaving no nucleotide overhang; these are referred to as "blunt ends," which is the type of cut performed by the Eco321 restriction enzyme. In the laboratory, we use restriction enzymes to cut plasmids for inserting DNA of interest. If a plasmid is cut with enzymes resulting in the formation of sticky ends, then the inserted DNA must also be cut with the same restriction enzymes to generate complementary sticky ends. The new DNA is incorporated into the plasmid by the formation of phosphodiester bonds linking the DNA to the plasmid. These phosphodiester bonds are created by the enzyme DNA ligase, which uses ATP to form these new bonds. Restriction enzymes are also used to cleave genomic DNA into smaller pieces. The frequency of cut-sites in DNA can be calculated and depends on the length of the recognition sequence. For a restriction enzyme with a 4 bp recognition sequence, and assuming that all bases are represented equally in the genome, the frequency of that sequence occurring in the genome is once every 4^4 bp (1 in 256 bp). If the restriction enzyme recognized a 6 bp sequence, the frequency of that sequence would be once every 4^6 bp (1 in 4,096 bp). Thus, restriction enzymes with longer recognition sequences cut less frequently than those with short recognition sequences. The DNA fragments produced by restriction digest are often separated using electrophoresis on an agarose gel. This enables the researcher to visualize the sizes of the fragments using a molecular ruler run alongside the digested DNA. Using the pGLO plasmid map in Figure 2, the predicted fragment lengths for a double digest using EcoRI and Eco321 would be 3694 bp and 1676 bp. * The purpose of this lab was that we used gel to verify that we have correct plasmids. The uncut DNA shows that you have to cut to see plasmid at the expected sites. If you don't cut the DNA before inserting it into the gel, it won't form bands where it is supposed to.

Tyrosinase Enzyme Kinetics What was the purpose of this graph and the table we used to make it?

Results from this data table will be used in the next sections to make a graph. The reaction catalyzed by tyrosinase converts DOPA (substrate) to dopachrome (product). This table can be used to determine the concentration of product formed by reading the absorbance of the reaction at 475 nm. We can use the standard curve generated to determine the micromoles (μmol) of dopachrome (product) formed. * "If my absorbance is 0.23, how much dopachrome formed? Oh, 6 micromoles!".

DNA Electrophoresis When cleaning up, what is important to remember about the gel rig?

Rinse the gel rig and electrophoresis box with dH2O. DO NOT DRY WITH PAPER TOWELS. The electrodes are easily broken. Allow to drip dry.

Isolation of Plasmid DNA by Mini Prep P2

SDS/sodium hydroxide (SDS/NaOH) + RNase A: Mixing bacteria with SDS/NaOH results in lysis of the bacterial cells. SDS denatures proteins while NaOH denatures both chromosomal and plasmid DNA. RNase A degrades RNA which could interfere with downstream applications. Our kit names this solution "P2."

Isolation of Plasmid DNA by Mini Prep Silica Membrane Technology

Silica Membrane Technology: We will utilize a commercially produced kit that uses a silica membrane to bind plasmid DNA. Once bound to the membrane, the DNA is washed with an ethanol-containing buffer (Buffer PE) which removes salts and SDS. The DNA is then eluted from the membrane using water or a buffer. We will use a buffer.

DNA Electrophoresis Differentiate the terms "dye" and "stain" in respect to this lab.

Stain: The Sybr Safe DNA stain helped use visualize the DNA after electrophoresis was performed. Dye: the xylene cyanol, bromophenol blue, and orange G dyes helped us visualize the electrophoresis progress.

Tyrosinase Enzyme Kinetics What factors of a cell's environment influence the activity of enzyme-catalyzed reactions?

Temperature, pH, presence of inhibitors. Because hundreds of reactions are simultaneously carried out in the living cell, it is difficult to study a single reaction in an intact cell. However, it is possible to extract enzymes from cells and thus study enzyme-controlled reactions in a test tube.

DNA Electrophoresis What does % agarose gel tell you about the separation of DNA?

The % agarose in the gel influences the separation of DNA fragments during electrophoresis. A higher % gel will be able to resolve smaller differences in the size of DNA fragments loaded. For instance, low % gels (0.3-0.5%) are used to separate large DNA molecules 20-60 kb, while high % gels (2%) are used to separate small DNA molecules 100-500 bp (0.1-0.5 kb).

DNA Electrophoresis Looking at a student's gel below (pictured), what advice would you give this student who was told that they must redo the gel because of its non-publishable quality. Assume the band sizes seen in lanes 2 and 3 are expected and correct.

The advice I would give the student would be to leave lanes 1 and 5 empty, rather than lanes 4 and 5. The reason that lane 1 is messed up is because it wasn't left empty. The only lanes that should be filled should be lanes 2,3, and 4 since they're in the middle and not on the edge.

Isolation of Plasmid DNA by Mini Prep What can absorbance at 260 nm and 280 nm tell you?

The amount of DNA isolated can be quickly determined using the spectrophotometer set at a UV absorbance of 260 nm. DNA has peak absorbance at 260 nm while proteins have peak absorbance at 280 nm. Use the following conversion factor to determine the concentration of DNA from the A260: 50 ng/μL of DNA has an absorbance of 1.0 at 260 nm. (50 ng/μL = 50 μg/mL) DNA concentration in ng/μL = A260 x 50 ng/μL x dilution factor Yield = Concentration x total eluted volume Further, evaluation of the A260/A280 ratio indicates how much protein contaminants are present in the DNA prep. A ratio of 1.8 is considered pure for DNA, and a lower ratio indicates the presence of proteins while a ratio above 1.8 indicates RNA contamination.

Bacterial Transformation What is the function of the bla gene?

The bla gene encodes Beta-lactamase, an enzyme that breaks down the antibiotic ampicillin, transformants expressing the bla gene can be selected by placing ampicillin in the growth medium.

Bacterial Transformation Transformation efficiency * How do you calculate it? * What factors affect transformation efficiency?

The efficiency of transformation is defined as the number of transformants (colonies) per mg of DNA. It is assumed that each round colony grew from a single transformed cell. Thus to calculate the transformation efficiency, one needs to know how much DNA was used (ng), what the total reaction volume was, how much was plated, and the number of colonies. With this information the following equation can be used: Transformation efficiency = (# colonies) / ((ng DNA/ total rxn. volume μL) x (volume plated μL)) The result should be written in scientific notation. Typically only 3-10% of the competent cells are truly competent, able to take up DNA. The quantity & quality of DNA used also affects the transformation efficiency. The number of transformants increases linearly with increased DNA added to the reaction up to 10 ng per 100 μL of competent cells. Using more than 10 ng of DNA only modestly increases the number of transformants obtained, or the transformation efficiency. The quality of DNA used for transformation is also important. Higher transformation efficiency is seen with supercoiled plasmid DNA as compared to plasmid DNA that has been nicked and is relaxed. * For the graph, it jumps up and then jumps back down. Once it gets to a certain ng the transformation efficiency drops. * Too much DNA can inhibit a transformation reaction.

DNA Electrophoresis How can DNA electrophoresis be used to determine the size of DNA fragments?

The migration distance of DNA fragments is proportional to the log10 of the size in base pairs of the DNA fragment. Hence, DNA electrophoresis can be used to determine the size of DNA fragments.

Bacterial Transformation What is the Multiple Cloning Site? Then list the abbreviations of the eight restriction enzymes that make up the MCS (begins with Scal) (list the names only, not the cut sites).

The multiple cloning site is a region containing restriction sites (Ndel, HindII, EcoRI, etc.), sequences that permit the insertion or deletion of a gene of interest. 1. ScaI 2. EcoRI 3. ScaI 4. KpnI 5. SmaI 6. BamHI 7. PstI 8. HindIII

Bacterial Transformation What is ori? * What is the length of the ori (in bp).

The origin of pGLO plasmid DNA replication (essential for making more copies of the plasmid). * 245 bp in length.

Tyrosinase Enzyme Kinetics Look up the structure of benzoic acid. How does the presence of benzoic acid affect the activity of tyrosinase and pyrocatechol? Based on your comparisons of the structures of pyrocatechol and benzoic acid, might you expect benzoic acid to be a competitive inhibitor? (Note that benzoic acid is a weak acid and will not damage the enzyme through direct chemical action.)

The presence of benzoic acid affects the activity of tyrosinase and pyrocatechol by acting as a competitive inhibitor. The structures of pyrocatechol and benzoic acid are very similar, allowing benzoic acid to mimic pyrocatechol (the actual substrate). By mimicking pyrocatechol, benzoic acid can then bind directly to the active site, blocking pyrocatechol from binding, and inhibiting/slowing down the activity of tyrosinase.

Immunoprecipitation To which region of the antibody is Protein A/G on the beads attracted? Be specific.

The recombinant protein will bind to the heavy chain of the antibodies.

DNA Electrophoresis How should the lid on the electrophoresis box be placed?

The red wire (+) electrode should be on the end oppositie from your wells. * Run to red. DNA has a negative charge and will migrate toward the (+) electrode (anode;red wire). * Run the gel until the dye is at the bottom. Do not run it for too long! Watch it!

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) Which side of the plastic plate should be facing you on the gel rig?

The short side of the plastic plate should be oriented towards the back of the rig, the longest plate faces you. *** Also, note the color coordination of the electrodes - red to red and black to black. Red = (+) electrode; "run to red". *** *** Do not force pipet into the well, this will separate the 2 plates holding the gel, allowing air to get between the gel and one of the plastic plates. Once this happens, the gel is ruined. *** *** Make sure there are no bubbles in your wells. ***

DNA Electrophoresis What is the size of the genome for λ DNA? What kind of genome is it?

The size of the genome for λ DNA is 48,502 bp, and is a linear dsDNA genome

DNA Electrophoresis Using the info in the background section of the manual, make a map of the cut sites and fragment sites for EcoRI on the λ DNA genome. * Given: EcoRI cleaves λ DNA at positions: 21,226; 26,104; 31,747; 39,168; & 44,972 bp. * What is the size of the genome for λ DNA? This might not be given.

The size of the genome for λ DNA is 48,502 bp, and is a linear dsDNA genome. DRAW TO SCALE (TRY YOUR BEST)

Bacterial Transformation What are the three basic steps of transformation?

The transformation procedure has 3 basic steps: (1) incubation of competent cells with DNA on ice; (2) heat shock to encourage the bacteria to take up DNA, & (3) a recovery phase where the bacteria are grown in nonselective media to allow expression of selection genes (antibiotic resistance genes on the plasmid DNA) before plating a small volume on selection media. During the first step (incubation) the competent cells are in a solution of CaCl2. The positive charge of the calcium ions neutralizes the negative charge of the phosphates in the bacterial cell membrane as well as the phosphates in the DNA. It is during this step that DNA becomes attached to the outside of the bacteria. Performing this step on ice helps to stabilize the cell membrane that has been rendered competent by CaCl2 treatment. In the second step (heat shock) the rapid transfer from ice to 42o C creates a draft between the outside of the cell which is warmed and the inside which is still cool from incubation on ice. This draft pulls the DNA inside the cell. After the heat shock step an additional step of incubating on ice helps to stabilize holes in the cell membrane created by CaCl2 and heat shock treatment. The final phase (recovery) allows cells time to copy the transformed plasmid and express genes encoded by the plasmid before placing the bacteria on selective media. Growth on selective media typically requires the action of proteins such as beta-lactamase (providing ampicillin-resistance) for the cells to survive. The recovery phase typically varies between 30 minutes and 2 hours at 37oC. * In recovery, bacteria are grown, allowing antibiotic-resistant genes to be expressed in order for the cells to survive against antibiotics. This antibiotic-resistant gene is a result of the transformation by the pGLO plasmid. Since the arabinose (selective media) has ampicillin (the antibiotic), only the transformed cells will survive.

DNA Electrophoresis What way should the gel be oriented?

The wells should be oriented so that they are on the same side as the black (-) electrode.

Tyrosinase Enzyme Kinetics Enzyme Inhibition

These reactions took place at optimal temps for enzyme activity. So, although the temps vary among enzyme, all of them are inhibited at temps where they should be the most active.

Tyrosinase Enzyme Kinetics Effect of Temperature

These reactions took place with their optimal substrates for enzyme activity. So, although the substrates vary among enzyme, all of them reacting to produce optimal enzyme activity at different temps.

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) How do you determine molecular weight using SDS-PAGE?

To determine the molecular weight of an unknown protein, a series of molecular weight standards are run in parallel with the unknown protein. When the gels are stained with a protein-binding dye, such as Coomassie blue, blue protein bands show up on the gel according to the molecular weight (MW) of the protein. If the log of MW vs. the relative mobility (Rm) of the protein standards is graphed, a straight line should be obtained which can be used as a standard curve. (It is helpful to produce a semi-log plot, with the molecular weights plotted on the log axis.) The molecular weight of an unknown protein can be extrapolated from the data on the standard curve. Protein standards are almost always run in Lane 1 of the gel for use in comparing the size of other protein samples run on the gel. The protein standards are commonly referred to as a "ladder" and usually come prestained so the progress of the gel can be monitored during electrophoresis. In this lab, Precision Plus Protein™ Dual Color Standards from Bio-Rad (# 161-0374), are used. It contains ten proteins of known size, with eight blue-stained bands and two pink reference bands.

Bacterial Transformation Background

Transformation of bacteria has become commonplace in the laboratory as a way to introduce DNA into bacteria. Transformation requires the use of bacteria that have been treated to render them competent. These bacteria are often referred to as competent cells, meaning that they are able to take up extracellular DNA. Competent cells have changes in their cell wall and membrane which allow DNA to enter the cell. Competent cells are created by treating E. coli with calcium chloride (CaCl2) on ice. The calcium ions interact with the cell membrane and cell wall, making them more permeable to DNA. It is important that cells be in the logarithmic phase of growth when treated with CaCl2 to obtain the best competent cells. Further, these cells must be kept on ice after treatment with CaCl2 or frozen immediately. Such cells should be used once thawed and not re-frozen as competence is lost with multiple freeze-thaw cycles. Escherichia coli (E. coli) is a commonly used bacterial species for transformation, and will be used for this lab exercise. In this lab we will introduce the plasmid pGLO into E. coli competent cells (see the plasmid map below). pGLO encodes green fluorescent protein (GFP) that when expressed will make the bacterial cells glow green under UV light. The GFP was originally isolated from the jellyfish, Aequorea victoria. Its expression on the pGLO plasmid is driven by an arabinose promoter. A promoter is the region upstream of a gene that controls the expression of a gene (the promoter for the lac operon in E. coli is a great example). As in all organisms, gene expression is carefully regulated to prevent wasteful production of proteins and to adapt to different environmental conditions. The bacterial genes encoding the enzymes needed to metabolize (break down) the simple sugar arabinose are controlled by an upstream promoter region that regulates the expression of these enzymes. These genes are only activated in the presence of arabinose (makes sense—why produce enzymes to break down arabinose if the sugar is not present in the environment). In the case of the pGLO plasmid, the genes for these arabinose breakdown enzymes have been replaced with GFP. In the presence of arabinose, the GFP gene is expressed and the bacterial colonies will fluoresce a brilliant green. In the absence of arabinose, GFP is not expressed. The pGLO plasmid also encodes an ampicillin resistance gene, which allows for direct selection of transformed bacteria (bacteria containing the plasmid). Plating the bacteria on LB agar containing ampicillin and arabinose allows growth of only bacteria containing the plasmid and expression of the green fluorescent protein. * Without arabinose, GFP won't be produced.

DNA Electrophoresis How can we estimate the size of DNA fragments we're separating? * No graph

Typically, when using agarose gel electrophoresis to separate DNA fragments and estimate their size, one lane of the gel will contain molecular weight markers (commonly referred to as a ladder). Molecular weight markers are available commercially. The ladder used for this lab is known as the 1kb ladder, because it contains DNA bands of increasing size beginning with 1kb (1 kilobase-pair, or 1000 base-pairs): Alternatively, molecular weight markers can be prepared by digesting λ DNA with restriction enzymes. In this lab we will run pre-cut Lambda (λ) DNA; it has been cut with the restriction endonucleases HindIII &/or EcoRI. Lambda DNA comes from the lambda bacteriophage (virus) that infects E. coli. It is a commonly used source of DNA in the molecular biology lab, including studies of DNA integration and DNA modification.

Tyrosinase Enzyme Kinetics What is the relationship between tyrosinase, tyrosine, and melanin? Hint: look up the structures of L-DOPA & melanin online.

Tyrosinase converts tyrosine -> L-DOPA[quinone] (addition of an -OH group to aromatic ring), and then L-DOPA is used to make melanin.

Tyrosinase Enzyme Kinetics What is tyrosinase?

Tyrosinase is a member of a group of enzymes that catalyze the oxidation of diphenols or monophenols. Biologically, tyrosinase is an enzyme found in melanocytes which catalyzes the first step in melanin production. Melanin gives skin, hair, and eyes their color. An example of the chemical reaction catalyzed by tyrosinase is shown. In this reaction, the substrate DOPA (3,4-dihydroxyphenylalanine) is converted by tyrosinase to form the product dopaquinone. The dopaquinone product rapidly changes to dopachrome, a colored product, via a non-enzymatic reaction. This reaction commonly occurs during the browning of fruits and vegetables when they are cut or bruised and exposed to oxygen in the air. A similar reaction occurs in human skin in the metabolic pathway that forms melanin from the amino acid tyrosine. Once dopaquinone is formed in melanocytes, a series of additional chemical reactions convert dopaquinone to melanin in the skin, hair follicles, the iris, and the retina. The purpose of this set of experiments will be to investigate various properties of the tyrosinase enzyme. This enzyme is capable of oxidizing a number of substrates with structures similar to that of DOPA (3,4-dihydroxyphenylalanine). The following compounds can also be oxidized by tyrosinase. Note that the three compounds are isomers since they have the same empirical formula, C6H6O2, but differ in the position of the hydroxyl groups.

Tyrosinase Enzyme Kinetics Effects of substrate concentration * Velocity versus [S] - Be able to obtain Km and Vmax from these graphs, either one.

Varying the amount of substrate present, while keeping the enzyme concentration constant, reveals many important features of enzyme-substrate interactions. * Curve should be hyperbolic. * The blank will contain enzyme only, while the test cuvette will contain enzyme + substrate. * The velocity was obtained by: (2 micromoles dopachrome)/(time in min) * Remember, when active sites are filled (Vmax), increase in [S] doesn't do anything (they're busy).

Immunoprecipitation * What is immunoprecipitation? How did we do this? * What is the "free antibody approach"? What is the "pre-immobilized approach"? Which approach is used in this lab?

We can use antibodies to isolate a specific protein from cell lysate. We isolated GFP (Green Fluorescent Protein) from bacterial lysate, which contained many thousands of different proteins. Immunoprecipitation is a powerful technique that harnesses the specificity of antibodies to isolate a particular protein from cells that have been lysed open ("cell lysate"). Cells from various organisms can be used as a source of proteins, from bacterial cells to mammalian cells. In this lab, we will use bacterial cells as our protein source. First, the protein of interest must be expressed by the cells. One can rely on natural levels of expression or stimulate the cells to express higher levels of a particular protein. Cells can also be transformed so that they carry a plasmid containing the gene sequence for the protein of interest. Assuming the gene is under the control of a strong promoter (or grown such that transcription is induced), the gene will be transcribed and translated and the resulting protein can be purified. In this lab, bacterial cells will be transformed with the pGLO plasmid. A colony from this transformation will be transferred into broth containing ampicillin as well as arabinose and grown in an overnight culture. The cells will be harvested and then lysed using a lysis buffer specifically designed to break down thick bacterial cell walls. The resulting lysate is then subjected to immunoprecipitation. Antibody against the specific protein of interest is added to the cell lysate and mixed for an hour. The antibody we are using is from Santa Cruz Biotech (Cat #sc-9996); it is a monoclonal IgG antibody that binds to GFP (Green Fluorescent Protein). After the antibody has incubated with the lysate for 45 minutes, agarose beads containing proteins A and G are added to the mixture and incubated for an additional 30 minutes. Protein A/G is a recombinant protein that combines the IgG-binding domains of Protein A and Protein G (Santa Cruz Biotech sc-2003). This recombinant protein will bind to the heavy chain of antibodies. The beads have a high affinity for all species of antibody and IgG subclasses. Additionally, the beads have been pre-blocked with BSA to reduce non-specific binding of the Ab. Following incubation with beads, the mixture is spundown and washed several times. The beads containing the antibody-antigen complex form a pellet at the bottom of the centrifuge tube. The washes are critical to remove proteins that have been trapped among the beads. After the final wash, the supernatant (which contains all non-GFP proteins) will be discarded. The beads are then mixed with an electrophoresis sample buffer, which contains SDS and β-mercaptoethanol to denature proteins and disrupt disulfide bonds as well as glycerol and bromophenol blue for tracking. A boiling step ensures that all the antibodies are released from the beads. The supernatant is analyzed by SDS-PAGE. The immunoprecipitation method used in this exercise is called the "free antibody approach" because we are using free antibody to first combine with Ag prior to adding beads. In contrast, the "pre-immobilized approach" uses antibodies that are already attached to beads. * There are two proteins on the beads. Protein A and Protein G. Having both of these proteins gives better binding to the heavy chain of the antibody. GFP is the antigen (what the antibody is binding to). * We should see heavy AND light chains of antibody AND GFP in SDS gel. *We used antibodies to isolate GFP proteins from bacterial cells that have been lysed. 1. We transformed the bacterial cells using a pGLO plasmid so that they would express GFP (our protein of interest). 2. A colony of the transformation was transferred to a broth containing ampicillin and arabinose to allow the colony to grow. 3. The cells were then harvested and lysed using a lysis buffer. 4. The lysate was subject to immunoprecipitation. 5. Antibodies against the specific protein of interest (GFP) was added (the antibody is a monoclonal IgG antibody that binds to GFP). This mixture was allowed to incubate. The antigen-binding complex of the antibody connected to the protein of interest. 6. Agarose beads containing protein A and protein G were added to the mixture and allowed to incubate. * Proteins A and G bind to the heavy chains of the antibodies. * The beads have been pre-blocked with BSA to reduce nonspecific binding of the antibody. 7. Mixture was spun down and washed several times to remove unwanted proteins (in supernatant, beads and everything important formed a pellet at the bottom of the tube). 8. Beads were mixed with an electrophoresis sample buffer containing SDS and β-ME to denature proteins and disrupt disulfide bonds, as well as glycerol and bromophenol blue for tracking. 9. Solution was boiled, ensuring antibodies were released from the beads. 10. Supernatant analyzed by SDS-PAGE (antibodies and GFP in supernatant).

Tyrosinase Enzyme Kinetics Effects of Chemicals that Modify Enzymes

We see reduced activity. The chemical disrupted the native enzyme structure.

Immunoprecipitation Since GFP is not normally expressed in E. coli, how did we ensure that there would be enough GFP from E. coli to isolate by immunoprecipitation?

We transformed E. coli with the pGLO plasmid to make sure there was enough GFP.

DNA Restriction Digest Know how to make a plasmid map. Predict the outcome of a successful digest. Draw a plasmid map and calculate the expected fragment sizes.

When creating your own plasmid map, remember that the first base pair is in the 12 o'clock position. The name and size of the plasmid should be written in the center, and the cut sites of interest are indicated with a line, the name of the enzyme, and the exact site at which the DNA will be cut. *** DRAW TO SCALE ***

DNA Restriction Digest Super Mix (AKA Mega Mix) * Know how to create one.

When preparing multiple digests, a Super Mix (SM) is first produced. This mixture contains the restriction enzymes, buffer, BSA, and water. The SM volume is slightly larger than what is ultimately needed, since small volumes can be lost during pipetting. Thus, in our case, we will produce a SM with a volume sufficient for 3 reactions, even though we only have 2 reactions. For these reactions, we will digest approximately 300 ng of DNA per reaction. A typical SM looks like this: The buffer is supplied as a 10X stock solution; once diluted in the SM tube, it provides the appropriate pH conditions that are optimal for enzyme activity. When designing a double digest, such as the one performed in this lab, it is critical that the buffer chosen is compatible with both enzymes. Restriction Enzyme suppliers have charts on their websites to assist in choosing the appropriate buffer; some suppliers also have "finder" functions that will find the optimal buffer for you (use Google to search for "Thermo Fisher double digest calculator"). Buffers are typically used at a final 1x concentration. However, when double digests are performed, you will occasionally be told to use a different final concentration (such as 2x) for optimal activity of both enzymes. Always double-check the appropriate buffer concentration before calculating the volumes for your digest. Finally, BSA (Bovine Serum Ovalbumin) stabilizes enzymes and is needed for particular enzymes to function optimally. We will not be using BSA for the double digest in this lab. Once complete, the SM is added to each reaction tube; the last component added will be the plasmid DNA obtained from the pGLO mini-prep. Details on preparing a Super Mix (SM): First, calculate how many µL of plasmid DNA is needed for 300 ng total. Using your new plasmid volume, determine the amount of water to have a total reaction of 10 µL (the volume of the restriction enzyme and buffer cannot be changed; only the water and plasmid volumes can be adjusted). To calculate how much SM goes into each tube, subtract the plasmid volume from 10. * Total volume is 10 µL, but once you add the 2 µL of loading dye, its 12 µL

SDS-PAGE (SDS-Polyacrylamine Gel Electrophoresis) How would you go about calculating the antilog of something?

You just do 10^x. x = whatever number the log is. * Ex: log(125) = 2.1 10^(2.1) = 125

Isolation of Plasmid DNA by Mini Prep While the general public often thinks of the term cloning in reference to whole organisms (like Dolly the sheep), for the molecular biologist, what's most often cloned is a _____.

gene or other small piece of DNA.