Novexr ecl western blotting detection reagents

Immunoblotting was performed to measure p38 MAPK activation levels by protein phosphorylation detection in the presence or absence of EGF. Cells were lysed in RIPA buffer, and then the samples were heated to 95°C for 5 min, separated on a 4%–12% Bis-Tris NuPAGE gel (Life Technologies) and transferred to nitrocellulose for immunoblotting. Immunoblots were incubated with primary, then secondary antibodies and bound antibody levels were detected with the Novex^R ECL Western blotting detection reagents (Life Technologies).

IP samples were processed for SDS-PAGE and Western blot as follows: beads were washed five times with the IP buffer described above, resuspended in 50 μl of sample loading buffer and separated on 4–12% NuPAGE gels (Life Technologies). Where no heating was necessary, samples were incubated with loading buffer for 30 min at room temperature under reducing and/or non-reducing conditions. The resolved proteins were electroblotted onto PVDF membranes (GE Healthcare) using the XCell II Blot Module (Life Technologies). Transfer was conducted constantly at 30 V for 1 h. Blots were incubated with appropriate primary and secondary antibodies and protein signals were detected with the Novex^R ECL Western blotting detection reagents (Life Technologies). Where protein phosphorylation levels were measured, specific anti-phosphorylation primary antibodies were used. Blots were then stripped using Restore Western Blot Stripping Buffer (Thermo Scientific/Pierce, Rockford, IL) and re-probed with the desired antibodies for total protein detection. The signal intensity of phosphorylated bands was normalized with that of the total protein and the average ratios were plotted with standard deviations.