Anti-RAB29 antibody [MJF-R30-124]

• IP

Unknown

Immunoprecipitation - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

RAB29 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate with ab256526 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256526 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : A549 (human lung carcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab256526 IP in A549 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab256526 in A549 whole cell lysate

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

Exposure time : 30 seconds

All lanes:

Immunoprecipitation - Anti-RAB29 antibody [MJF-R30-124] (ab256526)

Predicted band size: 23 kDa

false

• WB

Lab

Western blot - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

False colour image of Western blot : Anti-RAB29 antibody [MJF-R30-124] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab256526 was shown to bind specifically to RAB29. A band was observed at 23 kDa in wild-type A549 cell lysates with no signal observed at this size in RAB29 knockout cell line ab280040 (knockout cell lysate ab280099). To generate this image, wild-type and RAB29 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 1/20000 dilution.

All lanes:

Western blot - Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

RAB29 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human RAB29 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rab29-knockout-a549-cell-line-ab280040'>ab280040</a>)

Lane 2:

Western blot - Human RAB29 knockout A549 cell lysate (<a href='/en-us/products/cell-lysates/human-rab29-knockout-a549-cell-lysate-ab280099'>ab280099</a>)

Lane 3:

MCF-7 cell lysate at 20 µg

Lane 4:

Caco-2 cell lysate at 20 µg

Predicted band size: 23 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

This antibody reacts with an unidentifiable protein around 150 kDa. This image was kindly provided by Dr. Mark Cookson, NIH.

Blocking/Diluting buffer and concentration : 50% TBST, 50% Odyssey Blocking buffer

Exposure Time : 3 min

All lanes:

Western blot - Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/3000 dilution

Lane 1:

HEK293T cells (human embryonic kidney epithelial cell) transiently transfected with non-target siRNA control whole cell lysate at 15 µg

Lane 2:

HEK293T cells (human embryonic kidney epithelial cell) transiently transfected with siRNA against Rab29 whole cell lysate at 15 µg

Secondary

All lanes:

IR Dye 800CW Goat anti-rabbit IgG (#926-32211) at 1/10000 dilution

Predicted band size: 23 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

The lysates were kindly provided by Dr. Dario Alessi, University of Dundee.

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

Exposure Time : 3 min

All lanes:

Western blot - Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/1000 dilution

Lane 1:

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

Lane 2:

A549 (human lung carcinoma epithelial cell) Rab29 KO 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>)

Predicted band size: 23 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

Western blot : Rabbit Monoclonal[MJF-R30-124] to RAB29 ab256526 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 23 kDa in Wild-type U-87 MG ab278079 cell lysates with no signal observed at this size in RAB29 knockout U-87 MG ab306765 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG ab278079 at 20 µg

Lane 2:

RAB29 knockout U-87 MG ab306765 at 20 µg

Lane 3:

MCF7 at 20 µg

Lane 4:

Caco-2 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 23 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-RAB29 antibody [MJF-R30-124] (AB256526)

This antibody reacts with unidentifiable proteins around 30,75 and 150 kDa. This image was kindly provided by Dr. Mark Cookson, NIH.

Blocking/Diluting buffer and concentration : 50% TBST, 50% Odyssey Blocking buffer.

Exposure Time : 3 minutes.

All lanes:

Western blot - Anti-RAB29 antibody [MJF-R30-124] (ab256526) at 1/3000 dilution

Lane 1:

wildtype C57BL6 mouse lung tissue lysate at 15 µg

Lane 2:

Rab29 knock-out C57BL6 mouse lung tissue lysate at 15 µg

Secondary

All lanes:

IR Dye 800CW Goat anti-rabbit IgG (#926-32211) at 1/10000 dilution

Predicted band size: 23 kDa

Observed band size: 23 kDa

false