SDS-PAGE electrophoresis Materials: 1.5 M tris-HCl pH 8.8 1.5 M tris-HCl pH 6.8 40% acrylamide (29:1 acrylamide:bis-acrylamide) 10% sodium dodecyl sulfate (SDS) 20% ammonium persulfate isopropanol concentrated TEMED 5x loading dye (50% glycerol, 50 mM beta-mercaptoethanol (BME), 10 mM tris pH 8, 20% SDS] IX tris-glycine-SDS buffer 1) Decide what % acrylamide gel you want to run, given the size protein you wish 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 about 5-10 mL of acrylamide solution and the stack about 2 mL 3) Mix together your reagents for the body of the gel, adding the TEMED last 4) Use a serological pipette to fill the gel plates. 5) Apply ~1 mL isopropanol using a pipetteman and allow to polymerize, -20 mins. 6) Mixtogether the stack reagents, adding the TEMED last. 7) Dump the isopropanol and use a kimwipe to dry the top of the gel 8) 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 buffer 12) Carefully remove the comb and rinse the wells. 13) Load 7-10 uL protein ladder to the first lane 14) 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) - overnight 17) 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.

SDS-PAGE electrophoresis Materialsmestroupe/protocols/SDS-PAGEElectrophoresis.pdf · SDS-PAGE electrophoresis Materials: 1.5 M tris-HCl pH 8.8 1.5 M tris-HCl pH 6.8 40% acrylamide

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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.