重组Anti-Bak抗体[Y164] (ab32371)

Immunohistochemical analysis of paraffin-embedded fixed (A) Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet. (B) BAK1 knockout HeLa (ab265277) cell pellet staining Bak with ab32371 at 1/2000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Counter-staining used was hematoxylin.
Positive staining on (A) Wild-type HeLa cell pellet, no staining on (B) BAK1 knockout HeLa (ab265277) cell pellet.
The section was incubated with ab32371 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized BAK1 KO Hela (human BAK1 knockout cervical adenocarcinoma epithelial cell, Left) /Parental Hela (Right) cells labelling Bak with ab32371 at 1/500 dilution (0.1 µg)(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Lanes 1- 2: Merged signal (red and green). Green - ab32371 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32371 was shown to react with Bak in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265277 (knockout cell lysate ab257077) was used. Wild-type HeLa and BAK1 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. ab32371 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreatic carcinoma tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Lanes 1 - 3: Merged signal (red and green). Green - ab32371 observed at 25 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab32371 was shown to specifically recognize BAK in wild-type HAP1 cells. No band was observed when BAK knockout cells were examined. Wild-type and BAK knockout samples were subjected to SDS-PAGE. Unpurified ab32371 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

ab32371 (purified) at 1:20 dilution (2μg) immunoprecipitating Bak in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab32371 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32371 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human stomach tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemical analysis of Bak expression in paraffin embedded human stomach carcinoma, using 1/250 unpurified ab32371.

Bak was immunoprecipitated from HCT116 p53-/- cell line whole cell lysate with unpurified ab32371 at 1/100 dilution.

Western blot was performed from the immunoprecipitate using ab32371 at 1/2000 dilution.

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