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SDS Polyacrylamide Gel Electrophoresis

SDS-PAGE is an example of a MultiphasicBufferSystem for the resolution of proteins, however, proteins are pretreated by boiling with the detergent SDS (sodium dodecylsulfate) in the presence of reducing agent prior to loading. This causes the polypeptide chains to be unfolded and coated with an approximately uniform negative charge. Proteins are thereby separated primarily on the basis of their molecular weight.

Bio-Rad makes a popular SDS-page minigel apparatus for which there are gel recipies.

stacking gel

degas under vacuum before initiating polymerization

resolving gel

degas under vacuum before initiating polymerization

To initiate polymerization add APS (ammonium persulfate) and TEMED.


Reagents

4x Tris/SDS pH 6.8 stacking gel buffer
  1. dissolve 6.05 g Tris-base in 40 ml H2O
  2. adjust pH to 6.8 with 1 N HCl
  3. add H20 to 100 ml
  4. add 0.4 g SDS
store at room temperature

4x Tris/SDS pH 8.8 resolving gel buffer

  1. dissolve 91 g Tris-base in 300 ml H2O
  2. adjust to pH 8.8 with 1 N HCl
  3. add H2O to 500 ml
  4. add 2 g SDS
store at room temperature

5x SDS electrophoresis buffer

Do not adjust pH (should be 8.3). Store at room temperature.

6x SDS sample buffer

store in 1 ml aliquots at -20C


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