Anti-RAB35 antibody [MJF-R34-25] - BSA and Azide free

• WB

Supplier Data

Western blot - Anti-RAB35 antibody [MJF-R34-25] - BSA and Azide free (AB288576)

This data was developed using ab288576, the same antibody clone in a different buffer formulation.

ab288567 was shown to react with RAB35 in wild-type HAP1 cells in Western blot with loss of signal observed in a RAB35 knockout cell line. Wild-type HAP1 and RAB35 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab288567 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-RAB35 antibody [MJF-R34-25] (<a href='/en-us/products/primary-antibodies/rab35-antibody-mjf-r34-25-ab288567'>ab288567</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

RAB35 knock-out HAP1 lysate at 20 µg

false

• WB

Lab

Western blot - Anti-RAB35 antibody [MJF-R34-25] - BSA and Azide free (AB288576)

This data was developed using ab288567, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lysates/proteins at 20 μg per lane.

Performed under reducing conditions.

False colour image of Western blot : Anti-RAB35 antibody [MJF-34-R25] (ab288567) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab288567 was shown to bind specifically to RAB35. A band was observed at 25 kDa in wild-type A549 cell lysates with no signal observed at this size in RAB35 knockout cell lysates. To generate this image, wild-type and RAB35 knockout A549 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

Wild-type A549 and RAB35 knockout A549 lysates were kindly provided by Prof. Dario Alessi University of Dundee.

All lanes:

Western blot - Anti-RAB35 antibody [MJF-R34-25] (<a href='/en-us/products/primary-antibodies/rab35-antibody-mjf-r34-25-ab288567'>ab288567</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

RAB35 knockout A549 whole cell lysate at 20 µg

Lane 3:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 23 kDa

Observed band size: 25 kDa

false

• WB

Lab

Western blot - Anti-RAB35 antibody [MJF-R34-25] - BSA and Azide free (AB288576)

This data was developed using ab288567, the same antibody clone in a different buffer formulation.

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

This antibody has no cross-reactivity with other RAB family proteins

All lanes:

Western blot - Anti-RAB35 antibody [MJF-R34-25] (<a href='/en-us/products/primary-antibodies/rab35-antibody-mjf-r34-25-ab288567'>ab288567</a>) at 1/1000 dilution

Lane 1:

RAB35 full-length protein 10 ng

Lane 2:

RAB family protein homologous region 10 ng

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: 23 kDa

Observed band size: 25 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-RAB35 antibody [MJF-R34-25] - BSA and Azide free (AB288576)

This data was developed using ab288567, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-RAB35 antibody [MJF-R34-25] (<a href='/en-us/products/primary-antibodies/rab35-antibody-mjf-r34-25-ab288567'>ab288567</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg

Lane 3:

Mouse lung tissue lysate at 20 µg

Lane 4:

Mouse brain tissue lysate at 20 µg

Lane 5:

Rat brain tissue 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: 23 kDa

Observed band size: 25 kDa

false

Exposure time: 37s