Anti-Bax antibody [E63]

12 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bax antibody [E63] (ab32503)

  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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) was used as a secondary antibody at 1:500 dilution. Cytoplasmic staining on human lung carcinoma.

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  Immunoprecipitation - Anti-Bax antibody [E63] (ab32503)

  Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, 10μg
  Lane 2 (+): HeLa whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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. VeriBlot for IP Detection Reagent (HRP) 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: Immunoprecipitation - Anti-Bax antibody [E63] (ab32503)

  Predicted band size: 21 kDa

  Observed band size: 21 kDa

  Exposure time: 1s

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  Lanes 1 - 2: Merged signal (red and green). Green - ab32503 observed at 20 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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.

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

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: Bax knockout HAP1 cell lysate at 20 µg

  Predicted band size: 21 kDa

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  Lanes 1 - 2: Merged signal (red and green). Green - ab32503 observed at 20 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.

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

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: Bax knockout HAP1 cell lysate at 20 µg

  Predicted band size: 21 kDa

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  Lanes 1- 2: Merged signal (red and green). Green - ab32503 observed at 21 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (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 Human BAX knockout HeLa cell line ab255363 (knockout cell lysate Human BAX knockout HeLa 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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: Western blot - Anti-Bax antibody [E63] (ab32503) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: BAX knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human BAX knockout HeLa cell line (Human BAX knockout HeLa cell line ab255363)

  Performed under reducing conditions.

  Predicted band size: 21 kDa

  Observed band size: 21 kDa

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  Different batches of ab32503 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 0.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 18 kDa.

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

  Predicted band size: 21 kDa

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  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: Western blot - Anti-Bax antibody [E63] (ab32503)

  Predicted band size: 21 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bax antibody [E63] (ab32503)

  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.
  
  

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  Western blot - Anti-Bax antibody [E63] (ab32503)

  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.

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

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 2: Hep G2 (Human hepatocellular carcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 3: Jurkat (Human T cell leukemia T lymphocyte) Whole cell lysates at 20 µg

  Lane 4: A549 (Human lung carcinoma epithelial cell) Whole cell lysates at 20 µg

  Lane 5: C2C12 (Mouse myoblasts myoblast) Whole cell lysates at 20 µg

  Lane 6: C6 (Rat glial tumor glial cell) Whole cell lysates at 20 µg

  Lane 7: Mouse brain. Whole tissue lysate at 20 µg

  Lanes 8 and 10: Rat brain. Whole tissue lysate at 20 µg

  Lanes 9 and 11: Rat Spleen. Whole tissue lysate at 20 µg

  Secondary

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

  Predicted band size: 21 kDa

  Observed band size: 18 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bax antibody [E63] (ab32503)

  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.

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  Sandwich ELISA - Anti-Bax antibody [E63] (ab32503)

  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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bax antibody [E63] (ab32503)

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BAX with ab32503 at a concentration of 0.05 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  ab32503 Anti-Bax antibody [E63] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)