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STANDARD OPERATING PROCEDURE:

SDS-Polyacrylamide Gel Electrophoresis

(adapted from

Acrylamide is a suspected carcinogen and a known neurotoxin. Handle unpolymerized solutions of acrylamide with great care.

Wear a lab coat, gloves, and eye protection when working with acrylamide. Avoid any contact of acrylamide solutions with skin.Replace gloves if they become contaminated.

Pour gels in a fume hood when possible. Areas where gels are poured should be protected with a lab bench cover. Avoid splashes.

Avoid handling powdered acrylamide by purchasing premixed solutions (in its powder form, the monomer is extremely dangerous because the dust can easily become airborne and enter the respiratory system).

Health Hazards for Acrylamide:

Toxic! Ingestion may cause systemic poisoning. May cause drowsiness, tingling sensations, fatigue, weakness, stumbling, slurred speech, and shaking.May cause central and peripheral nervous system damage.

Severe intoxication may cause permanent nerve damage. May affect reproductive system and act as a teratogen.

Skin exposure may cause irritation and redness. Can be absorbed through the skin causing systemic poisoning; symptoms may parallel ingestion. For eye or skin exposure, flush with water for at least 15 minutes.

Refer to the MSDS for additional safety information on Acrylamide. Refer to EH&S fact sheet on electrophoresis.

Preparing Acrylamide Gels

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. 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.

Running the Gel

Use caution when operating electrophoresis units, as the combination of high voltage and exposed liquid can be hazardous. Never operate the unit without its cover in place. Turn off power supply and, be careful when plugging and unplugging the electrical cables (it’s safest to use one hand when doing this), and verify that the power supply is working properly.
Run the 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