Synthetic peptide within Human Bax aa 1-100 (N terminal). The exact sequence is proprietary.
A trial size is available to purchase for this antibody.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab32503 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
---|---|---|
IHC-P | 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. | |
WB | 1/1000 - 1/10000. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa). | |
IP | 1/100. | |
Sandwich ELISA | Use at an assay dependent concentration. Can be paired for Sandwich ELISA with Mouse monoclonal [2D2] to Bax (ab77566). |
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 (HRP) was used as the secondary antibody at 1/1000 dilution. Capture antibody was used at a 1/20 dilution. Blocking and diluting buffer used was 5% NFDM/TBST.
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.
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Bax knockout HAP1 cell lysate (20 µg)
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 when Bax knockout samples were used, 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: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Bax knockout HAP1 cell lysate (20 µg)
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 = 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 50oC). 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.
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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"