Lysis buffer could be selected based on the application. For example, immuno-precipitation experiment requires proteins in native form, whereas western blotting requires protein in denatured form. Based on the application, the buffer constituents vary, for example, SDS (ionic denaturing detergent) would be added in the buffer for western blotting while a non-ionic detergent such as Nonidet P-40 or Triton X-100 would be used for co-IPs.
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