SDS-PAGE: One-Dimensional Gel Electrophoresis of Proteins

Outline:

This one-dimensional gel electrophoresis of proteins is performed as denaturing (SDS) discontinous gel electrophoresis according to the Laemmli method. The concentration of the acrylamide gel depends on the protein size. The stacking gel is the upper part for loading the sample. Proteins are well lined up when they reach the lower gel (separating gel).

Supplies & Equipment:

• BioRad Mini-PROTEIN II
• 2 Erlenmeyer flasks (125 mL)

Reagents:

• 30% acrylamide
• "Upper Tris" (4 x Tris-Cl / SDS, pH6.8)
• "Lower Tris" (4 x Tris-Cl / SDS, pH8.8)
• 10 % APS
• TEMED
• ddH₂O

Procedure:

• Clean and assemble BioRad Mini-PROTEIN II apparatus. The following amounts are sufficient for two minigels.

• Prepare Separating Gel: In an Erlenmeyer flask, mix 30% acrylamide solution, 4 x Tris / SDS, pH 8.8, and H₂0. Add 10% APS and TEMED. Swirl gently to mix. Use immediately. Pour ~ 5 mm below where comb ends. Cover with H₂0. Polymerization is finished when the line between water and gel becomes visible and stable (i.e. does not change if gel inclined).

• Prepare Stacking Gel: In an Erlenmeyer flask, mix 30% acrylamide solution, 4 x Tris / SDS, pH 6.8, and H₂0. Add 10% APS and TEMED. Swirl gently to mix. Pour off covering water from separating gel. Use immediately. Insert clean (!) comb. Failure to form a firm gel usually indicates a problem with the APS, TEMED or both.

• Prepare samples, do not exceed 100 mg total protein. For 1 mm thick gels, the volume of a well is ~ 35 µL. For e.g. 20 µl total volume use this:

  • 14 µl sample
  • 5 µl 4 x sample loading buffer
  • 1 µl 2-mercaptoethanol

  Boil 5 min. before use. Load 20 µl per lane.

• Prepare protein standards (20 µl total volume):

  • 5 µl of 4 x sample loading buffer
  • 1 µl of protein standard marker
  • 1 µl 2-mercaptoethanol
  • 1 µl pyromin red solution
  • 12 µl H₂O

  Boil 5 min. before use. Load 10 µl per lane.

• Electrophoresis condition: BioRad Mini-PROTEIN II (2 gels); 200 V constant or 30 - 40 mA constant, in 1 x Running Buffer (= 250 mL 4 x Running Buffer, 10 mL of 10% SDS, 740 mL H₂O). Process gel further (Western Blotting).

Time Required: 2 hours

Notes:

• Poor or slow polymerization of acrylamide is most likely the result of an older solution of ammonium persulfate (APS) solution which has gone off. First try making a fresh stock from solid material. Netters have several quick tests to see if the APS is still good. One way is to put some in a test tube and add the correct volume of H20 to make 10 mg/ml. If you hear it snap, crackle, and pop, then it is still good. This is because some gas forms within the crystals when water is added to APS which causes them to pop. Another way is to prepare solution of NaI in water and add your APS to it. If the color changes to brown, the APS is still good. Opinions vary concerning how long APS is stable. Some netters store a 10% stock of APS at 4 degrees C for several weeks, while others make 10% APS and freeze single use aliquots at -20. Still others make just enough fresh solution for a single gel just before pouring one. If all tests for polymerization fail, the next thing to test is the solution of TEMED. Buy a fresh bottle and start over.

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