All lanes : Anti-Bax antibody [E63] (ab32503) 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 - ab32503 observed at 21 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32503 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. ab32503 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.

Purified ab32503 staining Bax in Human lung carcinoma tissue section by immmunohistochemistry (IHC-P- Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraffin and heat mediated antigen retrieval was performed using EDTA buffer (pH 9.0). Samples were incubated with primary antibody at 1:500 dilution. A goat anti-rabbit IgG H&L (HRP) (ab97051) was used as a secondary antibody at 1:500 dilution. Cytoplasmic staining on human lung carcinoma.

All lanes : Anti-Bax antibody [E63] (ab32503)

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 - ab32503 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 ab32503 and a competitor's top cited rabbit polyclonal antibody.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, 10μg
Lane 2 (+): HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32503 in HeLa whole cell lysate

Purified ab32503 immunoprecipitating Bax in HeLa lysates. For western blotting, the primary antibody used was purified ab32503 at 1/1000 dilution. Ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection at 1/1000 dilution. Capture antibody was used at a 1/20 dilution. Blocking and diluting buffer used was 5% NFDM/TBST.

All lanes : Anti-Bax antibody [E63] (ab32503) at 1/1000 dilution

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 - ab32503 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32503 was shown to recognize Bax in wild-type HAP1 cells, along with additional cross-reactive bands. Wild-type and Bax knockout samples were subjected to SDS-PAGE. ab32503 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

IHC image of ab32503 staining Bax in rat kidney formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32503, 1:250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Lane 1 = Bax protein (Tagged) (ab85157), 10 ng. Lane 2 = Extract of HeLa cells, 40 ug. Lane 3 = Extract of HepG2 cells, 40 ug. Lane 4 = Bax protein (Tagged) (ab85157), 10 ng. Lane 5 = Extract of HeLa cells, 40 ug. Lane 6 = Extract of HepG2 cells, 40 ug. SDS PAGE performed under reducing conditions (100mM DTT Sample heated at 50^oC). Primary : Lanes 1-3: Anti Bax antibody (ab77566) at 1 ug/mL. Lanes 4-6: Anti Bax antibody (ab32503) at 1:2000 dilution. Secondary : Lanes 1-3: Goat anti mouse IgG(H&L)-HRP at 1:10000. Lanes 4-6: Goat anti rabbit IgG(H&L)-HRP at 1:10000. Development: ECL with 2 min exposure. Blocking: in 5% Milk + PBS for 3 hours at RT. Primary antibody: in 5% Milk + PBS overnight at 4 C. Secondary antibody: in 5% Milk + PBS for 2 hour at RT. Predicted band size : Bax 21kDa and Bax (Tagged) 49 kDa. Observed band size : Bax 21kDa and Bax (Tagged) 49 kDa.

All lanes : Anti-Bax antibody [E63] (ab32503) at 1/2000 dilution (purified)

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 buffers: 5% NFDM/TBST

Exposure time 1~9 lanes 32 s; 10~11 lanes 3 min

Jurkat is negative reported by PMID: 15528359. Brain is low expressed reported by PMID: 27069530.

Immunohistochemical analysis of paraffin-embedded human lymph node using anti-Bax Rabbit Monoclonal Antibody (ab32503) at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Standard Curve for Bax (Analyte: Recombinant human Bax protein (tagged) ab85157) dilution range 1pg/ml to 1ug/ml using Capture Antibody Mouse monoclonal [2D2] to Bax - BSA and Azide free (ab77566) at 0.2ug/ml and Detector Antibody Rabbit monoclonal [E63] to Bax (ab32503) at 0.5ug/ml Concentration of ab32503 may vary from lot to lot; please use this curve as guideline.