Page 1 of 1 Performing SDS-PAGE CHEM 4581: Biochemistry Laboratory I Version: February 2008 GOALS One of the goals of this week’s lab is for students to learn how to pour, run, stain, and destain an SDS-PAGE gel. For the purpose of teaching, students will be given one or more protein samples in which to run on a gel. TA’s will be carefully watching student performance in order to correctly instruct students on the subtleties of performing this experiment. Students will hopefully develop their skills in performing SDS-PAGE for a direct application in next week’s lab. MATERIALS AND REAGENTS Mini-Protean III Gel Electrophoresis Apparatus Bio-Rad 1.5 M Tris-HCl buffer, pH 8.8 Students will prepare this buffer for themselves 0.5 M Tris-HCl buffer, pH 6.8 Students will prepare this buffer for themselves 10% Ammonium Persulfate Make fresh for each section 10% Sodium Dodecyl Sulfate 40% (w/v) Acrylamide:Bisacrylamide (35:1 molar ratio) N,N,N’,N’-Tetramethylethylenediamine (TEMED) Laemmli Sample Buffer Must add 2-mercaptoethanol to this buffer daily 10X Electrophoresis Running Buffer Must dilute to 1X before use Coomassie Stain Recycle stain solution Destain EXPERIMENTAL PROCEDURES 1. Prepare each of the Tris-HCl buffer solutions. These solutions will be stored and used in a subsequent laboratory session.  10-20 mL of 0.5M Tris-HCl, pH 6.8  20-30 mL of 1.5M Tris-HCl, pH 8.8 2. Clean and completely dry the glass plates, combs, and any other pertinent materials. 3. Place a short plate on top of a spacer plate. Insert both plates into the green casting frame on a flat surface. Be sure that the "legs" of the casting frame are down. Clamp the casting frame and check that the plates are level on the bottom. 4. Put the casting frame into the casting stand. 5. Combine all reagents (except the APS and TEMED) in the order listed in the table below for a 10% resolving gel. Be sure to carefully add the acrylamide mixture to your solution in the fume hood. Cover your 15-mL conical tube while transporting it back to your workbench. 6. When ready to pour the gel, quickly add the APS and TEMED, quickly mix using a Pasteur pipette, and transfer the resolving gel solution between the glass plates in the casting chamber to about 3/4 inch below the short plate.Page 2 of 2 7. A small layer of butanol can be added on top of the gel prior to polymerization to straighten the level of the gel and remove unwanted air bubbles that may be present. Butanol will not mix with the aqueous separating gel solution. Once the gel has polymerized, the butanol can be removed by absorption with filter paper. Rinse the top layer of the gel with dI water prior to pouring the stacking gel. PREPARATION OF SDS-PAGE GEL MIXTURE Reagents 5% Stacking Gel (Total volume = 2 mL) 10% Resolving Gel (Total Volume = 5 mL) Deionized Water 1.2 mL 2.38 BUFFER 0.5 mL of 0.5M Tris-HCl, pH 6.8 1.25 mL of 1.5M Tris-HCl, pH 8.8 10% SDS 20 μL 50 μL 40% Acrylamide:Bisacrylamide Caution: Neurotoxin – do not ingest 250 μL 1.25 mL 10% Ammonium Persulfate 20 μL 50 μL TEMED 10 μL 20 μL 8. Insert the well forming comb into the opening between the glass plates. 9. Combine all reagents except the APS and TEMED in the order listed in the table above for the 5% stacking gel. 10. When ready to pour the gel, quickly add the APS and TEMED, quickly mix using a Pasteur pipette, and transfer the stacking gel solution between the glass plates in the casting chamber. There should be no air bubbles underneath the comb when you finish pouring the stacking gel layer. 11. Place ~400 mL of tap water in a 600 mL beaker and leave on a hot plate to boil. (This can take 15 minutes or more.) 12. Dilute your protein samples appropriately with Laemmli Sample Buffer that contains 2-mercaptoethanol (LSB) to a final volume of ~30 μL. Usually you would combine 10 μL of the protein sample with 20 μL of LSB. For the purpose of this exercise, your TA will offer guidance. Prepare your samples in microcentrifuge tubes. 13. In a separate microcentrifuge tube, add 3 μL of the MW marker to 6 μL of Laemmli sample buffer with 2-mercaptoethanol. 14. With the caps tightly closed, boil the samples for 2-3 minutes to fully denature the proteins. Leave the samples at room temperature until ready to load onto the gel. 15. Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate facing the inside. (This step is important! It is possible to situate the electrode assembly in the exact opposite orientation. Electrophoresis will not occur if this mistake is made.) 16. Slide the electrode assembly (with the gel cassette) into the clamping frame. Press down on the electrode assembly while clamping the frame to secure the electrode assembly. This step is important to minimize potential leakage during the electrophoresis experiment. 17. Pour some 1X electrophoresis buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel. Clean the wells with a syringe and 22-gauge needle. Be careful not to stab the glass plates. 18. When all wells are sufficiently cleaned, slowly pipette ~15 μL of denatured sample or ~5 μL of the MW marker into each lane. A yellow guide can be placed on top of the electrode assembly to aid in loading the gel.Page 3 of 3 19. When the gel has been loaded, lower the clamping frame into the electrophoresis tank. 20. Fill the region outside of the frame with 1X electrophoresis buffer. 21. Cover the tank with the lid aligning the electrodes (black or red) appropriately. Connect the electrophoresis tank to the power supply. Allow the samples to run at 150-200 V until the dye front reaches the bottom of the gel. 22. Meanwhile, put about 20 mL of Coomassie stain into your gel-staining box. 23. When electrophoresis is complete, turn off the power supply, disassemble the apparatus, and transfer the gel to the stain in the staining box. Allow the gel to stain on a rocking platform for ~15 minutes. Meanwhile, start cleaning all relevant equipment. 24. At the end of the staining period, pour the stain back into the bottle. (It is so concentrated that it can be used multiple times before being discarded.) Immediately add some Destain solution to the gel to prevent it from drying. (Note: