Lab 3 - PAGE Guide F23.pdf

Polyacrylamide Gel Electrophoresis (SDS-PAGE) Our experiment continues today! In Lab 1 we discussed cell culture and you compared the morphology and viability of stressed and unstressed Ptk2 cells. Labs 2 through 4 are all about processing your cell lysate samples to compare the expression of a par...

Polyacrylamide Gel Electrophoresis (SDS-PAGE) Our experiment continues today! In Lab 1 we discussed cell culture and you compared the morphology and viability of stressed and unstressed Ptk2 cells. Labs 2 through 4 are all about processing your cell lysate samples to compare the expression of a particular protein (either Hsp27 or p53) in your control and treated samples. i.e., do the Ptk2 cells make more or less of a specific protein in response to the stressor (either acid shock or oxidative stress)? In Lab 2 we discussed protein extraction and you did a Bradford Assay to determine the total protein concentration in cell lysates from stressed and unstressed cells. Now that you know the protein concentrations in your two samples, we can proceed to diluting them to the same concentration and separating the proteins via SDS-PAGE. This will set us up to do western blotting in Lab 4, where we can finally assess if the protein expression has changed! Learning Outcomes: By the time this lab is complete, you should be able to: • • Describe protein gel electrophoresis theory. Demonstrate pipetting proficiency. BEFORE Lab 3, you must: • • • • • Carefully read the rest of this document and print it. (We are back to ‘no devices’ this week!) Watch the “Protein Electrophoresis” video on Brightspace. Complete Table 2. (yes, complete before the lab!) Complete a flowchart for Lab 3. Complete the pre-lab quiz on Brightspace. (Heads-up, the quiz includes questions from your previous labs!) McCarville, Garant and Tatar (2023/2024) Electrophoresis: Electrophoresis is a common method for separating charged molecules (such as DNA or proteins) in an electric field. The molecules are driven through a cross-linked matrix, referred to as a “gel”, which acts like a molecular sieve, allowing differential migration of molecules based on size. Proteins are generally electrophoresed through gels made of polyacrylamide (hence the name “PAGE”, polyacrylamide gel electrophoresis) and are separated according to molecular weight. DNA is generally electrophoresed through agarose gels and is separated according to number of base pairs. Small molecules fit easily through the pores in the gel and migrate rapidly. Larger molecules become entangled in the gel matrix and therefore move slowly. Protein Preparation: Prior to electrophoresis, the cell lysates are mixed with a Sample Buffer. Although the exact composition of sample buffers varies, most contain the negatively charged detergent sodium dodecyl sulfate (SDS). SDS disrupts protein folding, which causes the proteins to denature and become rod-shaped – meaning that the movement of the protein through the gel does not depend on the protein’s shape. SDS also coats the polypeptides with negative charges to increase the speed at which the proteins will migrate towards the positively charged electrode. (Note that most proteins do already carry a negative charge in buffers with a basic pH, but SDS will also mask any existing charges so that all the proteins become negatively charged). Sample buffer may also contain a reducing agent, such as b-mercaptoethanol or DTT, to break the strong disulfide bonds within the protein. (SDS does not break disulfide bonds!) Sample buffer is normally prepared as a 2x stock solution and is mixed in equal volumes with the protein sample. To ensure that the proteins have fully denatured before running them through a gel, the samples are boiled for a few minutes. SDS-PAGE vs Native-PAGE: The gels we use today contain SDS, which ensures the proteins are denatured. However, in some scenarios it may be desirable to keep the native structure of the cell lysate proteins. Native PAGE omits denaturing chemicals in the sample buffer and the gel. This will conserve interactions between polypeptide subunits and can provide extra information regarding protein structure and folding! Molecular Weight Standards: If proteins of known molecular weights (purchased from biotech companies and called “standards”, “ladders”, or “markers”) are run alongside the protein samples in the same gel, their migration distances can be plotted as a function of log molecular weight to produce a straight line, called a standard curve. The researcher can compare the distances migrated by sample proteins to the graph to accurately estimate their molecular weights. There are various molecular weight ranges that can be purchased; the example shown here includes 11 proteins ranging from 200 kDa to 6 kDa. This particular marker set is convenient to use because protein has been pre-stained. That means we can follow the migration of the marker proteins in real time as the gel is running! Fun! McCarville, Garant and Tatar (2023/2024) Task 1: Prepare the protein samples: Over the summer we grew Ptk2 cells and set up an experiment by stressing cells with either vinegar (acid shock) or H2O2 (oxidative stress). We also prepared unstressed cells as a negative control for each treatment. After incubating the cells in the appropriate reagent for 15 hr, we lysed the cells in RIPA buffer and collected the cell lysates. Once the protein concentrations of the lysates were determined (like what you did last week in Lab #2!) they were mixed with 2x Sample buffer (containing SDS, mercaptoethanol, glycerol, and tracking dye), aliquoted into microcentrifuge tubes and frozen. These are the samples you will receive today. Acid Shock Experiment: • Treatment: These cells were stressed with vinegar. The lysate was aliquoted into green tubes labeled “V”. • Neg. Control: These cells received water. The lysate was aliquoted into clear tubes with a green dot labeled “C”. Oxidative Stress Experiment: • Treatment: These cells were stressed with H2O2. The lysate was aliquoted into red tubes labeled “H2O2”. • Neg. Control: These cells received water. The lysate was aliquoted into clear tubes with a red dot labeled “C”. Last lab you ran a Bradford assay to determine the protein concentrations in your samples. In case you were off by a little bit, the actual protein concentrations are as follows: Experiment Acid Shock Oxidative Stress Cell Lysate: Treatment (“V”) Negative Control (“C”) Treatment (“H2O2”) Negative Control (“C”) Cell lysate concentration (µg/µl) determined by a Bradford assay: 10.98 9.34 8.14 7.12 Cell lysate concentration (µg/µl) after dilution with 2x Sample Buffer: (i.e. the samples you get today!) 5.49 4.67 4.07 3.56 During today’s lab, you will run your cell lysates through a gel. To compare protein expression between the samples, you need to make sure you run the same total number of micrograms (µg) of protein in each lane of the gel. You will now dilute your cell lysates further with 1x Sample Buffer so that the stressed (i.e. Treatment) and unstressed (i.e. negative control) samples are both at the same concentration of 1.0 µg/µl. That way, when we load our samples into the gel, we are loading the same amount of protein and can make a fair comparison. This ensures that any observed differences in protein expression are due to experimental conditions and not because you loaded more or less of a particular sample. BEFORE arriving at the lab, complete Table 2 below by calculating how to prepare 50 µl of a 1.0 µg/µl solution of each cell lysate (Treatment and Negative Control, after dilution of 2x Sample buffer) that you will use today. Based on your previous labs, will you use the lysates from the acid-shock or oxidative stress experiment? _______________ • • • • C1 – Concentration of the cell lysate provided (in table above) V1 – Your unknown value (how much of the cell lysate you need to remove from the tube) C2 – 1.0 µg/µl (the final concentration you want) V2 – 50 µl (the final volume you want) Table 2 Cell Lysate Treatment Negative Control Volume of provided cell lysate (µl) Volume of 1x Sample Buffer (µl) McCarville, Garant and Tatar (2023/2024) In the Lab! 1. Find your cell lysates and the molecular weight standard (labeled “M”). Double-check that you have the right ones (acid shock treatment + negative control or oxidative stress treatment + negative control). 2. Check with your TA or Instructor that you did your Table 2 calculations correctly. Using those values, dilute your cell lysates with 1x Sample Buffer in new microcentrifuge tubes. Make sure to label the two new tubes and remember to press only to the first stop before sucking liquid up into the micropipettor. Note: the molecular weight marker has been premixed with sample buffer and is ready to use. 3. If you have any of the original cell lysates leftover, please pass those tubes to your TA to put back into the freezer. 4. Place the tubes of newly diluted protein into a rack in the hot water bath (don’t forget the tiny reusable clamps). Heat for 5 min to ensure that all proteins are denatured. No need to boil the marker. Start preparing your gel while you wait. 5. After 5 min, remove the tubes from the hot water bath. To eliminate bubbles and bring the solution back to the bottom of the tube, centrifuge at room temperature for ~10 sec. You should also spin the “M” tube containing the marker proteins. Task 2: Position the Acrylamide Gel in the Electrophoresis Tank: The tank will accommodate two gels, but each pair will only use one gel today. 1. Wearing gloves, place a black clamp (labelled “L”) into the left chamber of the tank. 2. Pour Running Buffer into the chamber with your clamp, so that it is level with the silver electrode (i.e., the silver bar that runs horizontally near the top of each chamber). 3. The gels that we use in Cell Bio are pre-made and have an acrylamide concentration of 12%. The gel is delicate (only 1.0 mm thick) and is positioned between two clear plastic plates. At a sink, use scissors to cut open the packaging and remove the gel. Dispose of the packaging into a garbage. Remove the plastic comb and throw it in the garbage immediately. Use a KimWipe to gently wipe away any stringy acrylamide bits that may be remaining on the plastic plates. 4. Hold the gel upright and peek at the well tails to ensure they are standing upright. If not, use the dissecting tool to correct them. Peel off the white strip at the bottom and toss into the garbage immediately. 5. Drop the gel into the chamber in front of the black clamp. Position the gel so that the lower slanted plastic plate faces you and the front of the tank. It is normal (and necessary!) for the running buffer to go into the wells. McCarville, Garant and Tatar (2023/2024) 6. Pull the lever on the clamp forward to lock the gel into place. Make sure the running buffer is slightly above the silver electrode (top it up with additional running buffer if necessary). You are now ready to load! Fun!! Task 3: Load Marker and Cell Lysates into the Gel: Using a P20, load your gel based on the pattern below. Make sure to position the pipette tip so that you can see it inside the marked well. This is easier if you are eye-level with your sample. 1X Sam ple B uff er Ma rke r (“ M” ) Ne gat ive Co ntr ol ( T re “C” atm ) en t (S tre sse 1X d) Sam ple Bu ffe r 1X Sam ple Bu ffe r Ma rke r (“ M” ) Ne gat ive Co ntr ol ( T re “C” atm ) en t (S tre 1X sse Sam d) ple Bu ffe r When you press the plunger, the glycerol in the sample buffer will help to make your sample sink into the bottom of the well. Use a different tip for each sample to avoid contamination. Remember to keep your thumb steady so that you do not suck up your sample or spill it into a neighbouring well. Remember, the purpose of using the gel to separate proteins is to prepare for western blotting. To demonstrate an authentic experimental approach, you will load the samples in duplicate so that you can eventually probe for two different proteins during Lab 4. Everyone will probe for tubulin, but you will have your choice of apoptotic-associated Hsp27 or p53 proteins too! Will the stressor cause their protein expression to change? We will find out in Lab 4 – Exciting! J 1. Load 5 µl of the marker, and 15 µl of each sample. (Be sure that you AND your partner each get to try loading a few lanes!) You will also load 15 µl of 1x Sample Buffer in the “empty” lanes to help the samples run evenly. 2. Fit the lid onto the unit. It will only go on one way. Attach the electrodes to a power supply. Set the power supply to 110 mAmps and 200 volts. When you first turn on the current it is normal for the gel to run at only about 100 volts. This will shift as the gel runs. The tracking dye in the sample buffer allows us to follow the progress of electrophoresis. This charged blue dye migrates through the gel just ahead of the smallest proteins. McCarville, Garant and Tatar (2023/2024) Task 4: Remove the Gel from Between the Plastic Plates 1. When the tracking dye is close to the bottom of the gel (or when you can discern most of the prestained marker bands), stop the unit, disconnect the electrodes, and remove the lid. 2. Put on a pair of gloves and carry the gel unit to the sink. Carefully pour out the buffer. (Don’t let the gel and clamp fall into the sink!) 3. Back at your bench, unlock the clamp and remove the gel. Use the gel knife (the tool that looks like a putty knife) to separate the two plastic plates to expose the gel. Immediately pour a few ml of running buffer on the gel so it doesn’t dry out. Use the knife to remove the well tails and the bottom part of the gel below the blue tracking dye line. (You do not need those parts as there are no proteins there!) (H) 4. Dip the gel knife into some running buffer and carefully transfer the gel to the plastic dish with running buffer in it. Task 5: Transfer Proteins from the Gel onto Nitrocellulose: In preparation for Lab 4, when you will be performing a western blot, the proteins in the gel must be transferred to a piece of nitrocellulose membrane. This is achieved by stacking the gel on top of the nitrocellulose membrane and using a Bio-Rad Trans-Blot Turbo Machine to apply a current. This moves the negatively charged proteins out of the gel and onto the nitrocellulose membrane. The instrument will transfer the proteins in just 7 minutes! 1. Bring your gel in the small container to the side bench. 2. In the drawer, layer in order: filter paper, nitrocellulose, gel, and a second filter paper (see below). Once you have placed the gel on the nitrocellulose, use the tiny roller to ensure there are no bubbles in the stack (once again, bubbles are bad!). In this case, the bubble will prevent efficient transfer of the proteins. 3. Two gels can fit in one tray. The negative electrode, i.e. the “lid” of the tray, is positioned on top, locked into place, and any excess buffer can be drained out. 4. Slide the drawer into the Turbo-blot machine and select ‘Run’! It will take 7 minutes to transfer. McCarville, Garant and Tatar (2023/2024) 5. While waiting, find a small square plastic dish and use tape and a sharpie to label the lid with your lab section, bench number and initials. 6. Pour a small amount of TBS-Tween (found on the side bench) into the dish. 7. When the transfer is complete, slide the drawer out and bring it to the station near the front of the room by the sink, where we will remove the lid. 8. The top filter paper and the gel can be thrown away. 9. Put the nitrocellulose into your labelled dish and make sure it is submerged in the TBS-Tween. Place the dish into the fridge in a pile with the other membranes from your lab section. Woo hoo! The nitrocellulose is now ready for western blotting in the next lab!! FYI… If you were interested in immediately seeing alllllllllll the proteins present in your cell lysate, you could stain the gel with Coomassie Blue. However, this has limited usefulness, as it is almost impossible to know which band corresponds to any particular protein of interest. (Picture Right - Two lanes of cell lysate proteins in a Coomassie-stained gel. Notice that there are sooooooooo many proteins that it is almost blurry, and difficult to distinguish one band from another. Note: many proteins can have the same molecular weight, so each band could actually be hundreds of different proteins that ended up at the same spot in the gel. Amazing!) socratic.org Before you leave the lab today: • • • • Try to make your bench look the same as it did when you started the lab! NOTE: we will keep any remnants of protein that you have from the original tube. Give the tubes to your TA to put in the freezer. Remove your gloves and wash your hands. Complete the In-Lab Worksheet and submit it before you leave. McCarville, Garant and Tatar (2023/2024)