Other product details
Western Blot antibody
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. Proteins were immunoprecipitated by incubating lysates with anti-TRPM8 antibody ab85617 (10 µg/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, and added to the samples for 2h at 4°C. Immunoprecipitates were washed three times, and heated at 60° for 20 minutes in SDS-sample buffer.
Western Blot Assay
Immunoprecipitates and whole cell lysates were separated on 8% polyacrylamide gels, followed by transfer onto PVDF membranes.
The membrane was blocked in 5% milk-TBST solution overnight at 4°C, and then incubated with 1:1000 anti-TRPM8 ab85617 for 2h at room temperature. Washing was repeated three times in TBST buffer for 10 minutes each. Then, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody for 1 hour, followed by three washes in TBST (10 min each). Finally, the membrane was processed for chemiluminescence detection using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.).
NOTE: The above mentioned experimental protocols were also performed using another Abcam anti-TRPM8 antibody, ab3243 (purchased months earlier), which proved to be efficient.
Miss. Larisa Maier
Submitted May 15 2013