Use of RIPA buffer or NP-40 buffer with sufficient freshly added phosphatase inhibitors is needed for extracting phosphorylated proteins. Addition of phosphatase inhibitors is mandatory for protecting the phosphorylated group as during the cell lysis, release of phosphatase enzyme (such as acid/alkaline phosphatase, etc) would cleave the phosphorylated group in protein. Further, addition of non-ionic detergent (NP-40, Triton X-100, etc) would desirable as they would increase the solubility of non-polar in soluble proteins.  

The modified RIPA buffer 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
• 1mM Proteinase Inhibitor (Phenylmethylsulfonyl fluoride and/or dithiothreitol)
• 1mM of Sodium orthovanadate (Na3VO4)
• 10mM of Sodium Fluoride (NaF)
• 5mM Sodium pyrophosphate (Na4P2O7)
• 10mM β-glycerophosphate.

In the same way add the phosphatase inhibitors in NP-40 buffer, which consists of:

• 1% Nonidet P-40 (NP-40) or Triton X-100
• 150 mM NaCl, 50mM Tris-HCl (pH 8)
• 1mM Proteinase Inhibitor (Phenylmethylsulfonyl fluoride and/or dithiothreitol)
• 1mM of Sodium orthovanadate (Na3VO4), 10mM of Sodium Fluoride (NaF)
• 5mM Sodium pyrophosphate (Na4P2O7)
• 10mM β-glycerophosphate.