Recombinant Anti-RAB10 antibody [MJF-R23] (ab237703)

Lanes 1 - 4: Merged signal (red and green). Green - ab237703 observed at 25 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab237703 was shown to recognize RAB10 in wild-type A549 cells as signal was lost at the expected MW in RAB10 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and RAB10 knockout samples were subjected to SDS-PAGE. Ab237703 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% saponin permeabilized RAB10 KO A549 (RAB10 knockout human lung carcinoma epithelial cell) (ab261868) labelling RAB10 (red) with ab237703 at 0.04 μg/ml, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution. The nuclear counter stain is DAPI (blue).

Confocal image showing cytoplasmic staining in wild-type A549 cell line, and no staining in RAB10 KO A549 cell line. Images were acquired on the Perkin Elmer® Operetta HCA and a maximum intensity projection of 7 confocal planes is shown for the representative images. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% saponin permeabilized A549 wild-type and knock-out cells labeling RAB10 (red) with ab237703 at 0.5 μg/ml, followed by anti-Rabbit secondary at 1/1000 dilution. The nuclear counter stain is DAPI (blue). 

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

The WT and Rab10 KO A549 lysates were kindly provided by our collaborator Dr. Dario Alessi, University of Dundee.

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

RAB10 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma cell line) whole cell lysate using ab237703 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab237703 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 dilution was used for detection.

Lane 1: A549 whole cell lysate 10μg (input)
Lane 2: ab237703 IP in A549 whole cell lysate.
Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab237703 in A549 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (human breast adenocarcinoma cell line) cells labeling RAB10 (green) with ab237703 at 1/500 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing cytoplasmic staining in MCF7 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells labeling RAB10 (green) with ab237703 at 1/500 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing cytoplasmic staining in A549 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) cells labeling RAB10 with ab237703 at 1/600 dilution (red) compared with the rabbit monoclonal IgG (ab172730) isotype control (black) and an unlabelled control (cells without incubation with primary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077)at 1/2000 dilution was used as the secondary antibody. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% saponin permeabilized A549 wild-type and knock-out cells labeling RAB10 (red) with ab237703 at 0.5 μg/ml, followed by anti-Rabbit secondary at 1/1000 dilution. The nuclear counter stain is DAPI (blue).