Application
Immunoprecipitation
Immuno-precipitation step
Protein A
Sample
Human Cell lysate - whole cell (Hek cells transfected with human TRPM8)
Specification
Hek cells transfected with human TRPM8
Total protein in input
800 µg
Other product details
Concentration
6 µg/mg lysate
Incubation time
20 hour(s) and 0 minute(s) · Temperature: 4°C
Western Blot antibody
Name
Abcam antibody:Anti-TRPM8 antibody
Additional data
Additional Notes
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
MISS. Larisa Maier
Verified customer
投稿 Sep 17 2013