All lanes : Anti-RAB10 antibody [MJF-R23] (ab237703) at 1/1000 dilution

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : RAB10 knockout A549 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 22 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?

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 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). 

All lanes : Anti-RAB10 antibody [MJF-R23] (ab237703) at 1/1000 dilution

Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell line) whole cell lysate
Lane 2 : Rab10 knockout A549 whole cell lysate
Lane 3 : HCT 116 (human colorectal carcinoma epithelial cell line) whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 5 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 7 : C6 (rat glial tumor glial cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 10 seconds

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.

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.

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).