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What is the best way to make your own lysis buffer for protein isolation?
Posted December 9, 2020

Answer

The best way to make own lysis buffer is to optimize the ingredient concentration to best suit the requirement. 

The best buffer for maintaining the native form of a protein is a non-denaturing buffer. It consists of: 

  • 1% Nonidet P-40 (NP-40) or Triton X-100
  • 150 mM NaCl
  • 50mM Tris-HCl (pH 8)
  • 2mM EDTA (Optional)
  • Proteinase Inhibitor (Phenylmethylsulfonyl fluoride and/or dithiothreitol) 1mM

The best buffer for obtaining denatured protein is SDS lysis buffer. It consists of:

  • 2% SDS
  • 50mM Tris-HCl (pH 8)
  • 10mM EDTA
  • 10% Glycerol
  • Proteinase Inhibitor (Phenylmethylsulfonyl fluoride and/or dithiothreitol) 1mM

For obtaining sub-cellular proteins (mitochondrial or nuclear proteins), the best buffer would be radioimmunoprecipitation assay (RIPA) buffer. It consists of

  • 150 mM NaCl
  • 1% NP-40 or Triton X-100
  • 50mM Tris-HCl (pH 8.0)
  • 0.1% SDS
  • 0.5% sodium deoxycholate
  • Proteinase Inhibitor (Phenylmethylsulfonyl fluoride and/or dithiothreitol) 1mM

 

Possible variations in the buffer constituents include varying detergent concentration, increasing salt concentration, or changing the detergents (such as saponin, CHAPS, digitonin, etc.,) in the protocol


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