Cells were washed in ice-cold PBS solution and lysed in HEPES-CHAPS buffer (2% CHAPS in 50 mM Hepes, 200 mM NaCl pH 7.5), and a mixture of protease inhibitors (Roche) for 30 min on ice. Immunoprecipitation was performed by incubation with anti-TRPM8 antibody ab3243 (6 ´g/mg lysate) at 4¯C, overnight. Protein A-Sepharose beads (Sigma) were washed and equilibrated in 50 mM Hepes, 200 mM NaCl pH 7.5 buffer.
Western Blot Assay:
Immunoprecipitates and whole cell lysates were separated on 8% polyacrylamide gels, followed by transfer onto PVDF membranes.
The membrane was processed for chemiluminescence detection using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.).
Image: First lane: IP, Second lane: whole cell lysate.
Miss. Larisa Maier
Submitted Sep 17 2013
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