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

• 3.9% acrylamide overall
  
  • 37.5 : 1 acrylamide : bisacrylamide
    
• Tris/SDS pH 6.8 buffer
  

degas under vacuum before initiating polymerization

resolving gel

• overall acrylamide percentage can vary from 4% to 20%
  
  • 20 : 1 acrylamide : bisacrylamide provides the best resolution
    
• Tris/SDS pH 8.8 buffer
  

degas under vacuum before initiating polymerization

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

• add 1/100^th volume 10% APS (store 10% APS at 4C for up to two weeks)
  
• add 1/2500^th volume TEMED for fast polymerization, 1/5000^th volume for slow
  

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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

• 15.1 g Tris-base
  
• 72.0 g glycine
  
• 5.0 g SDS
  
• H₂O to 1000 ml
  

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

6x SDS sample buffer

• 7 ml 4x Tris/SDS pH 6.8 stacking gel buffer
  
• 3.0 ml glycerol
  
• 1 g SDS
  
• 0.93 g DTT (dithio-threitol)
  
• 1.2 mg bromophenol blue
  
• add H₂O to 10 ml
  

store in 1 ml aliquots at -20C

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Comments

• boil samples in SDS sample buffer for 1 minute, cool on ice and load
  
• Since this is a non-equilibrium method, never pre-run the gel. Doing so will destroy the stacking effect.
  
• Proteins will not migrate faster than the ionic front. Since tracking dye typically runs with the ionic front there is nothing to be gained by stopping the gel before the tracking dye has run off.