SDS-PAGE electrophoresis

Materials:

1.5 M tris-HCl pH 8.81.5 M tris-HCl pH 6.840% acrylamide (29:1 acrylamide:bis-acrylamide)10% sodium dodecyl sulfate (SDS)20% ammonium persulfateisopropanolconcentrated TEMED5x loading dye (50% glycerol, 50 mM beta-mercaptoethanol (BME), 10 mM tris pH8, 20% SDS]IX tris-glycine-SDS buffer

1) Decide what % acrylamide gel you want to run, given the size protein youwish to separate.

20-50 kDa 12 %50-100 kDa 10%100+ 7.5 %

2) Set up clean glass plates on the pouring rig knowing that each gel takes about5-10 mL of acrylamide solution and the stack about 2 mL

3) Mix together your reagents for the body of the gel, adding the TEMED last4) Use a serological pipette to fill the gel plates.5) Apply ~1 mL isopropanol using a pipetteman and allow to polymerize, -20

mins.6) Mix together the stack reagents, adding the TEMED last.7) Dump the isopropanol and use a kimwipe to dry the top of the gel8) Apply the stack and insert the comb. Allow to polymerize.9) Prepare your sample to IX loading dye. Mix, then boil for 5 mins.10) When the gel is ready, assemble the running rig. Make sure the small plate

faces the inner gasket and the dam is tight.11] Fill the rig with IX tris-glycine running buffer12) Carefully remove the comb and rinse the wells.13) Load 7-10 uL protein ladder to the first lane14) Add 20-40 uL (depending on well size) to the lanes.15) Run the gel at 180-200 V for 30-45 minutes.16) Remove from plates and stain in IX coomassie blue stain for SOmins (heat

10 s in microwave) - overnight17) Rinse in water, destain in IX destain (add a kim wipe to make it better

and/or 10 sec. in the microwave))18) Image over a light box.