SDS-PAGE Gradient Gel (3-15%)

-The Han Lab Ref: Laemmli,U.K. (1970) Nature, 227: 680-685 System: Hoefer SE 600 model (18cmX16cm) Wash and Setup of gel plates 1. Clean glass plates with soap and water and rinse in deionized water and dry. 2. Wash plates with 95% ethanol and dry with paper towel 3. Put together plates, spacers and clamp assemblies. Do it on flat surface to ensure everything is level. 4. Place glass plates on stand. Pouring of separating gel 1. Setup stir plate on jack so the top of stir plate is 24cm high. Put gradient maker on plate. 2. Mark two 50ml tubes for 3 and 15%. 3. Get out buffers needed to make separating gel. 4. Turn on stir plate and make up 3 and 15% solutions according to recipe. 5. With mixing bar parallel to ground pour 15% in the inside chamber and 3% to the outside chamber with no flow going to plates. 6. Start flow to plates and the turn mixing bar up 90 degrees to allow flow from outside (3%) to inside (15%). 7. Let it flow until it is 12cm up the plate. Dont move gels until polymerized! 8. Rinse gradient maker and tubing and blow out air. 9. Add butanol to top of poured gels. 10. Allow gel to polymerize for 45 minutes. It is polymerized if there is an interface between butanol and gel. 11. Pour off butanol and wash with deionized water. 12. Place combs in top. Rinse combs with ethanol first. 13. Prepare stack solution and pour into top. 14. Remove air bubble by tapping and sliding combs. 15. Let gel polymerize for 45 minutes and pull out combs. Gently pull combs straight up and out. 16. Gently rinse wells with deionized water and remove excess water in wells with syringe. 17. Load samples in wells and cover gently with running buffer. 18. Put on top buffer chamber and fill above wire with running buffer. Check for leaks. 19. Place in bottom buffer chamber and pour in running buffer even with top buffer tank. 20. Run gel overnight at 60 volts for a total of about 1000 volt hours. Let the dye run off the bottom completely. If protein is less that 25kd run the dye front to very bottom of plate but not off the gel to insure you dont lose the sample. Gel Recipes: Separating Gel: (2 gels) 3%12%15% F.S. dd Water19.3ml10.3ml7.3ml Sep. Gel buffer7.5ml7.5ml7.5ml Acrylamide3.0ml12.0ml15.0ml TEMED11ul11ul11ul 10% APS180ul180ul180ul ?Acrylimide is Boehringer Mannheim Protein Gel Mix 37.5:1 # 1685 821 ?TEMED is Boehringer Mannheim #100 139

Stacking Gel: 2 gels4 gels F.S. dd Water13.3ml26.6ml

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SDS-PAGE Gradient Gel (3-15%)

-The Han Lab Stacking gel buffer5.0ml10.0ml Acrylamide2.3ml4.6ml TEMED15ul30ul 10% APS150ul300ul

Make 5X LSB [Stock] Buffer [Final] for 10ml 30% SDS 15% 5.0ml 2M Sucrose 0.575M 2.88ml 2M Tris-HCl pH 6.8 0.325M 1.63ml ??Mercapto EtOH 5% 0.5ml - Bromophenol Blue 0.002% 0.5mg Can store at room temperature**** Dont boil samples when adding 5X LSB******* * Add 5X LSB to sample at 5X!! Should not be diluted to 1X.

Buffers Separating Gel Buffer: (1.5M Tris-HCL , 0.4% SDS) ?Add 90 grams of Tris to 500ml of dd water and pH to 8.7 with HCl. ?Add 2grams of SDS and stir in until dissolved. ?Filter through a 0.45um filter and store at room temperature. Stacking Gel Buffer: (0.5M Tris-HCl, 0.4% SDS) ?Add 30 grams of Tris to 500ml of dd water and pH to 6.8 with HCl. ?Add 2grams of SDS and stir in until dissolved. ?Filter through a 0.45um filter and store at room temperature. * Check pH of both Stacking and Separating gel buffers regularly due to changes in pH will cause gels to run poorly. Usually see poor separation of pre-stained MWs. 5X Electrophoresis Running Buffer: (25mM Tris, 192mM Glycine, 0.1% SDS) ?Add 60.5 grams of Tris, 288 grams of glycine and 20 grams of SDS to 4 liters of dd Water. No need to pH. Should be close to 8.3. Store at room temp. ?Dilute 1:5 with buffer: water to use. (The end)

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