Anti-eIF4A2 抗体 (ab31218)

Western blot: Anti-EIF4A2 antibody (ab31218) staining at 1 ug/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab31218 was shown to bind specifically to EIF4A2. A band was observed at 40-50 kDa in wild-type A549 cell lysates with no signal observed at this size in EIF4A2 knockout cell line. To generate this image, wild-type and EIF4A2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

eIF4A2 was immunoprecipitated using 0.5mg Mouse skeletal muscle whole tissue extract, 5µg of Rabbit polyclonal to eIF4A2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse skeletal muscle whole tissue extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31218.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 47kDa: eIF4A2 .