Purpose

To describe the formulation of both reducing and non-reducing 5X SDS-PAGE sample buffer.

References

Polyacrylamide gel electrophoresis in SDS containing buffers using multiphasic buffer systems: Properties of the stack, valid Rf measurement and optimized procedure. Anal. Biochem. (1978), 78:459

Materials

  • RMS-177 Sodium Dodecyl Sulfate (SDS)
  • RMS-110 Tris
  • RMS-182 Glycerol
  • SOP-142 Formulation of 10% Bromophenyl Blue
  • RMS-184 Dithiothreitol (DTT)
  • RMS-111 Hydrochloric Acid (HCl), 37%
  • SOP-136 Formulation of 50% NaOH.

Equipment

  • Stir plate
  • Tube Rocker (Nutator #421105 or equivalent)
  • pH Meter and pH standards

 

Procedure

  1. Make 1M Tris pH 6.8 by adding 12.1 g Tris to 100 ml DI water.  Stir to dissolve.
  2. Standardize pH meter to pH 4 and 7.
  3. pH solution to 6.8 using 37% HCl.  If pH is overshot, raise pH with a 1:10 dilution of 50% NaOH/DI.
          For 5X REDUCING buffer: Add 5.2 ml 1M Tris pH 6.8 to a 1 gram vial of DTT.  Swirl to dissolve.
          For 5X NON-REDUCING buffer: Add 5.8 ml 1M Tris pH 6.8 to 50 ml disposable centrifuge tube.
  4. Add 1.3 g SDS.  Rock to dissolve.
  5. Add 6.5 ml Glycerol and 130 µl 10% Bromophenyl Blue. Rock to mix at least 30 minutes.
  6. Aliquot to screw cap tubes ( 24 x 0.5 ml) and store at -20°C.

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