Anti-RAB8A antibody [MJF-R22] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

This data was developed using ab237702, the same antibody clone in a different buffer formulation.

ab237702 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 ab237702 at 1/250 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.

• IP

Supplier Data

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

This data was developed using ab237702, the same antibody clone in a different buffer formulation.

Immunoprecipitation of RAB8A in HeLa cells. Lysates were prepared and immunoprecipitation was performed using 2 ug of ab237702 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.

• IP

Supplier Data

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

RAB8A was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell line) whole cell lysate using ab237702 at 1/30. Western blot was performed from the immunoprecipitate using ab237702 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysate 10μg (Input).
Lane 2 : ab237702 IP in A459 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab237702 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 (ab237702).

All lanes:

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] (<a href='/ko/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>)

Predicted band size: 24 kDa

Observed band size: 24 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

RAB8A was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate using ab237702 at 1/30. Western blot was performed from the immunoprecipitate using ab237702 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10μg (Input).
Lane 2 : ab237702 IP in NIH/3T3 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab237702 in NIH/3T3 whole cell lysate.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237702).

All lanes:

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] (<a href='/ko/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>)

Predicted band size: 24 kDa

Observed band size: 24 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

RAB8A was immunoprecipitated from varying amounts of MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate with 1 μg of ab237702 per IP test. Western blot was perfromed from the immunoprecipitate using ab237702 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/2000 dilution.

Lane 1 : MEF whole cell lysate (input).
Lane 2-7 : ab237702 IP in different amounts of MEF lysates from 25 μg to 800 μg whole cell lysate.
Lane 8 : Pre-immune IgG instead of ab237702 in MEF whole cell lysate.

Blocking buffer and concentration : 5% NFDM/TBST.
Diluting Buffer and concentration : 5% BSA/TBST.

Exposure time : 10 seconds.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237702).

All lanes:

Immunoprecipitation - Anti-RAB8A antibody [MJF-R22] (<a href='/ko/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>)

Predicted band size: 24 kDa

false

• WB

Supplier Data

Western blot - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

This data was developed using ab237702, the same antibody clone in a different buffer formulation.

ab237702 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 237702HeLa and RAB8A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab237702 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] (<a href='/ko/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>) 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='/ko/products/cell-lines/human-rab8a-knockout-hela-cell-line-ab264993'>ab264993</a>)

false

• WB

Supplier Data

Western blot - Anti-RAB8A antibody [MJF-R22] - BSA and Azide free (AB238651)

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

Exposure time : 10 seconds

The band around 50 kDa is unspecific band.

The WT and Rab8A KO A549 lysates were kindly provided by our collaborator Dr. Dario Alessi, University of Dundee.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab237702).

All lanes:

Western blot - Anti-RAB8A antibody [MJF-R22] (<a href='/ko/products/primary-antibodies/rab8a-antibody-mjf-r22-ab237702'>ab237702</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

Rab8A knockout A549 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

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

Lane 6:

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

Secondary

All lanes:

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

Predicted band size: 24 kDa

Observed band size: 24 kDa

false

Exposure time: 10s