Recombinant Anti-RAB35 antibody [MJF-R34-25] (ab288567)

Blocking and diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-RAB35 antibody [MJF-34-R25] (ab288567) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab288567 was shown to bind specifically to RAB35. A band was observed at 25 kDa in wild-type A549 cell lysates with no signal observed at this size in RAB35 knockout cell lysates. To generate this image, wild-type and RAB35 knockout A549 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 1/10000 dilution.

Wild-type A549 and RAB35 knockout A549 lysates were kindly provided by Prof. Dario Alessi University of Dundee.

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Blocking and diluting buffer and concentration: 5% NFDM/TBST

This antibody has no cross-reactivity with other RAB family proteins