WB analysis was performed as previously reported [12 (link)]. Briefly, cells were lysed in RIPA buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA pH 8, 10 mM Tris-HCl pH 8) containing 1% protease inhibitor cocktail (Roche). Cell extract was centrifuged for 15 min at 4 °C and heated for 5 min at 95 °C; 30 μg of total protein extract was subjected to SDS-polyacrylamide gel electrophoresis, blotted on nitrocellulose membrane (Bio-Rad) and incubated ON with the specific antibodies. Protein expression was detected by ECL chemiluminescence method (Bio-Rad).
Other organizations :
University of Campania "Luigi Vanvitelli", The Netherlands Cancer Institute, Ospedale Santa Maria, Biogem, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
Cell lysis in RIPA buffer containing 1% protease inhibitor cocktail
Centrifugation of cell extract for 15 min at 4 °C
Heating of cell extract for 5 min at 95 °C
SDS-polyacrylamide gel electrophoresis of 30 μg total protein extract
Blotting on nitrocellulose membrane
Incubation with specific antibodies overnight
controls
Positive control: Not specified
Negative control: Not specified
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to
get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
