Anti-RAB8A antibody [MJF-R22-79-3]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

ab241061 was shown to react with RAB8A in wild-type HeLa cells in immunocytochemistry with loss of signal observed in RAB8A knockout cell line ab264993. 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 ab241061 at 1/600 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 ug/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).

This data was 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

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Immunocytochemistry/ Immunofluorescence - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells labeling RAB8A with ab241061 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in A549 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling RAB8A with ab241061 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human bladder cancer (PMID : 14581456). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling RAB8A with ab241061 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human breast (PMID : 14581456). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunofluorescent analysis of 3% paraformaldehyde-fixed, 0.1% Saponin permeabilized A549 (human lung carcinoma cell line) and RAB8A knock-out A549 cells labeling RAB8A with ab241061 at 1/100 dilution.

This image is kindly provided by our collaborator Dr. Dario Alessi, University of Dundee.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) cell line labeling RAB8A with ab241061 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) cell line labeling RAB8A with ab241061 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling RAB8A with ab241061 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in HeLa cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Unknown

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

RAB8A was immunoprecipitated from 0.35 mg of A549 (human lung carcinoma cell line) whole cell lysate with ab241061 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab241061 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : A549 whole cell lysate 10 μg (Input).

Lane 2 : ab241061 IP in A549 whole cell lysate .

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab241061 in A549 whole cell lysate .

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 8 seconds.

All lanes:

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22-79-3] (ab241061)

Predicted band size: 24 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Immunoprecipitation of RAB8A in HeLa cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab241061 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was 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.

• WB

Supplier Data

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Lanes 1-4 : Merged signal (red and green). Green - ab241061 observed at 24 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab241061 Anti-RAB8A antibody [MJF-R22-79-3] was shown to specifically react with RAB8A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264993 (knockout cell lysate ab257195) was used. Wild-type and RAB8A knockout samples were subjected to SDS-PAGE. ab ab241061 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (ab241061) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAB8A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RAB8A knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rab8a-knockout-hela-cell-line-ab264993'>ab264993</a>)

Lane 3:

HCT116 cell lysate at 20 µg

Lane 4:

Human brain tissue lysate at 20 µg

Predicted band size: 24 kDa

Observed band size: 24 kDa

false

• WB

Unknown

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Lanes 1-4 : Merged signal (red and green). Green - ab241061 observed at 24 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab241061 Anti-RAB8A antibody [MJF-R22-79-3] was shown to specifically react with RAB8A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264993 (knockout cell lysate ab257195) was used. Wild-type and RAB8A knockout samples were subjected to SDS-PAGE. ab ab241061 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (ab241061) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAB8A knockout HeLa cell lysate at 20 µg

Lane 3:

HCT116 cell lysate at 20 µg

Lane 4:

Human brain tissue lysate at 20 µg

Predicted band size: 24 kDa

Observed band size: 24 kDa

false

• WB

Supplier Data

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

Exposure time : 7.75 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID : 15673612).

All lanes:

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (ab241061) at 1/1000 dilution

Lane 1:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

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

Predicted band size: 24 kDa

false

• WB

Supplier Data

Western blot - Anti-RAB8A antibody [MJF-R22-79-3] (AB241061)

ab241061 was shown to react with RAB8A in wild-type HeLa cells in Western blot with loss of signal observed in RAB8A knockout cell line ab264993. Wild-type HeLa and RAB8A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab241061 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was 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-RAB8A antibody [MJF-R22-79-3] (ab241061) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 30 µg

Lane 2:

RAB8A knock-out HeLa lysate at 30 µg

Lane 2:

Western blot - Human RAB8A knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rab8a-knockout-hela-cell-line-ab264993'>ab264993</a>)

false