Preparation of whole cell extracts and immunoblotting were carried out as described previously (12 (link), 27 (link), 63 (link)). Briefly, cells were harvested and lysed in a lysis buffer [10 mM tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% Na-deoxycholate supplemented with complete protease inhibitors] (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1 from Sigma-Aldrich). After thorough mixing and incubation at 4°C for 2 hours, lysates were then centrifuged at 12,000g at 4°C for 20 min. Supernatants were collected and stored in aliquots at −80°C. Immunoblots were carried out following standard procedures, and immunoreactivity was detected using enhanced chemiluminescence reaction (170-5061, Bio-Rad) under ChemiDoc MP System (Bio-Rad).
Protocol detail
Whole Cell Extract Preparation and Immunoblotting
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Buffer
Cell extracts
Cells
Chemiluminescence
Deoxycholate
Diagnostics
Immunoblots
Inhibitors
Nacl
Np 40
Phosphatase
Phosphatase inhibitor 1
Protease inhibitors
Tris
Corresponding Organization : Indian Association for the Cultivation of Science
Other organizations : Indian Institute of Science Education and Research Pune, Indian Institute of Technology Gandhinagar
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Variable analysis
independent variables
- Independent variables not explicitly mentioned.
- Dependent variables not explicitly mentioned.
- 10 mM tris-HCl (pH 8)
- 150 mM NaCl
- 0.1% SDS
- 1% NP-40
- 0.5% Na-deoxycholate
- Complete protease inhibitors (Roche Diagnostics, Indianapolis, IN)
- Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1 from Sigma-Aldrich)
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