Anti-RAB10 antibody [MJF-R23] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using ab237703, the same antibody clone in a different buffer formulation.

ab237703 was shown to react with RAB10 in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a RAB10 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab237703 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 ?g/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using the same antibody clone in a different buffer formulation (ab237703). 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.

• WB

Lab

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using ab237703, the same antibody clone in a different buffer formulation.

ab237703 was shown to react with RAB10 in wild-type HAP1 cells in Western blot with loss of signal observed in a RAB10 knockout cell line. Wild-type HAP1 and RAB10 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab237703 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-RAB10 antibody [MJF-R23] (<a href='/en-us/products/primary-antibodies/rab10-antibody-mjf-r23-ab237703'>ab237703</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 40 µg

Lane 2:

RAB10 knock-out HAP1 lysate at 40 µg

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% saponin permeabilized Human 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).

Image courtesy of Dr. Dario Alessi from University of Dundee

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

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).

Image courtesy of Dr. Dario Alessi from University of Dundee

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

• IP

Supplier Data

Immunoprecipitation - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703).

All lanes:

Immunoprecipitation - Anti-RAB10 antibody [MJF-R23] (<a href='/en-us/products/primary-antibodies/rab10-antibody-mjf-r23-ab237703'>ab237703</a>)

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using ab237703, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-RAB10 antibody [MJF-R23] (<a href='/en-us/products/primary-antibodies/rab10-antibody-mjf-r23-ab237703'>ab237703</a>) at 1/10000 dilution

All lanes:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 180s

• WB

Unknown

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237703)

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.

All lanes:

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (ab238655) at 1/1000 dilution

Lane 1:

Wild-type A549 (human lung carcinoma epithelial cell line) whole cell lysate at 20 µg

Lane 2:

Rab10 knockout A549 whole cell lysate at 20 µg

Lane 3:

HeLa (Human cervix adenocarcinoma epithelial cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Lane 6:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg

Lane 7:

C6 (rat glial tumor glial cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

• WB

Unknown

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (AB238655)

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

All lanes:

Western blot - Anti-RAB10 antibody [MJF-R23] - BSA and Azide free (ab238655) at 1/1000 dilution

Lane 1:

Wild-type A549 whole cell lysate at 20 µg

Lane 2:

RAB10 knockout A549 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 22 kDa

false