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Western blot analysis and immunoprecipitation Total cells lysates for immunoblotting or immunoprecipitation were lysed with buffer (0.1%Triton-100, 50 mM Tris-Cl, pH 7.5, 0.1% NP-40, 150 mM NaCl, 0.1 M EDTA) supplemented with a cocktail of phosphate and proteinase inhibitors for 30 min at 4 C

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The freeze-thaw process is brutal on peptide chains, causing them to fracture and become useless

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