Standard SDS-polyacrylamide gel electrophoresis Protocol

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SDS-Polyacrylamide Gel Electrophoresis Method :

Standard SDS-polyacrylamide gel electrophoresis (Laemmli)--gel preparation. Volumes given are sufficient for small (8 cm X 10 cm X 1.5 mm) gel format (10 ml of monomer). Scale up volumes as needed.

1. Pour the Separating Gel
Set up your gel apparatus, prepare separating gel monomer. Add TEMED just prior to pouring gel (I "pour" the gels using a pasteur pipet and a rubber bulb). Allow to polymerize before adding stacking gel by overlaying gently with water or n-butanol. With higher % gels, one can immediately pour the stacking gel on the unpolymerized separating gel. Be careful not to mix the two layers.

Separating Gels, in 0.375 M Tris, pH 8.8
7% 10% 12% 15%
distilled H2O 5.1 ml 4.1 ml 3.4 ml 2.4 ml
1.5 M Tris-HCl,
pH 8.8 2.5 ml 2.5 ml 2.5 ml 2.5 ml

20% (w/v) SDS
0.05 ml 0.05 ml 0.05 ml 0.05 ml
Acrylamide/Bis-acrylamide
(30%/0.8% w/v) 2.3 ml 3.3 ml 4.0 ml 5.0 ml

10% (w/v) APS
ammonium
persulfate 0.05 ml 0.05 ml 0.05 ml 0.05 ml
TEMED 0.005 ml 0.005 ml 0.005 ml 0.005 ml

Total 10.005 ml 10.005 ml 10.005 ml 10.005 ml
monomer


2. Pour the Stacking Gel

After the separating gel has polymerized, decant the overlay, prepare the stacking monomer, add the TEMED, and pour. Insert the comb and allow to polymerize completely before running.

Stacking Gels, 4.0% gel, 0.125 M Tris, pH 6.8
distilled H2O 3.075 ml
0.5 M Tris-HCl, pH 6.8 1.25 ml
20% (w/v) SDS 0.025 ml
Acrylamide/Bis-acrylamide
(30%/0.8% w/v) 0.67 ml
10% (w/v) ammonium persulfate 0.025 ml
TEMED 0.005 ml
Total Stack monomer 5.05 ml

For best results:

1. Make ammonium persulfate solution fresh daily.

2. Degas solutions before adding TEMED for 15 min at room temperature.

3. Running the gel

I usually run my gels at constant current, 25-50 mA, depending on gel size. Here's the recipe for 5X SDS-PAGE running buffer. Dilute to 1X before use.

5X Running Buffer, pH 8.3 (1 liter)
Tris Base 15 g
Glycine 72 g
SDS 5 g
distilled water to 1 liter

Store at room temperature until use.

4. Laemmli Sample buffer

Dilute samples at least 1:4 with sample buffer, heat at 95 C for 4 minutes prior to loading.
Sample Buffer (8 ml)
Distilled water 4.0 ml
0.5 M Tris-HCl 1.0 ml
Glycerol 0.8 ml
10% SDS 1.6 ml
beta-mercaptoethanol 0.4 ml
0.05% (w/v)
bromophenol blue 0.2 ml

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For more information see:

SDS PAGE Gel Electrophoresis