Anti-IRAKM 抗体 [Y278] - BSA and Azide free (ab228417)

False colour image of Western blot: Anti-IRAKM antibody [Y278] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32394 was shown to bind specifically to IRAKM. A band was observed at 68 kDa in wild-type THP-1 cell lysates with no signal observed at this size in IRAK3 knockout cell line ab281629 (knockout cell lysate ab282979). To generate this image, wild-type and IRAK3 knockout THP-1 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: IRAKM knockout HAP1 cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab32394 observed at 69 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32394 was shown to specifically react with IRAKM when IRAKM knockout samples were used. Wild-type and IRAKM knockout samples were subjected to SDS-PAGE. ab32394 and ab8245 (loading control to GAPDH) diluted to 1/5000 and 1/10000 respectively were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32394).