All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BAX knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 21 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab182734 observed at 21 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab182734 was shown to react with Bax in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255363 (knockout cell lysate ab263841) was used. Wild-type HeLa and BAX knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab182734 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BAX knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HT29 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 21 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab182734 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab182734 was shown to specifically react with BAX when BAX knockout samples were used. Wild-type and BAX knockout samples were subjected to SDS-PAGE. Ab182734 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : HuT-78 (Human Sezary syndrome cell line) whole cell lysate
Lane 4 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 6 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 7 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 8 : L-929 (Mouse connective tissue fibroblast cell line) whole cell lysate
Lane 9 : C2C12 (Mouse myoblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 21 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Negative control:

Jurkat doesn't express Bax. (PMID: 9583678).

All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/1000 dilution

Lane 1 : Human kidney lysate
Lane 2 : Human spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 21 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

This product reacts with a 15 kD isoform (PMID: 7607685).

All lanes : Anti-Bax antibody [EPR18284] (ab182734)

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Bax knockout HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 21 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab182734 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa or ab18058, observed at 130 kDa.

This western blot image is a comparison between ab182734 and a competitor's top cited rabbit polyclonal antibody.

All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat kidney lysate
Lane 7 : Rat spleen lysate
Lane 8 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 9 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 10 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 21 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1, 2 and 3: 3 seconds; Lane 4: 2 seconds; Lane 5: 3 seconds; Lane 6 and 7: 2 seconds; Lane 8, 9 and 10: 1 second.

This product reacts with a 15 kD isoform (PMID: 7607685).

All lanes : Anti-Bax antibody [EPR18284] (ab182734) at 1/2000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell). Whole cell lysates
Lane 2 : Hep G2 (Human hepatocellular carcinoma epithelial cell). Whole cell lysates
Lane 3 : Jurkat (Human T cell leukemia T lymphocyte) Whole cell lysates
Lane 4 : A549 (Human lung carcinoma epithelial cell) Whole cell lysates
Lane 5 : C2C12 (Mouse myoblasts myoblast) Whole cell lysates
Lane 6 : C6 (Rat glial tumor glial cell) Whole cell lysates
Lane 7 : Mouse brain. Whole tissue lysate
Lanes 8 & 10 : Rat brain. Whole tissue lysate
Lanes 9 & 11 : Rat Spleen. Whole tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 21 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and diluting 5% NFDM/TBST

Exposure time 1~9 lanes 24 s; 10~11 lanes 3 min

Bax was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab182734 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab182734 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

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

Lane 2: ab182734 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab182734 in HeLa whole cell lysate.

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

Exposure time: 10 seconds.