Recombinant Anti-BAT3/BAG-6 antibody [EPR9223] (ab137076)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: BAT3/BAG-6 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab137076 observed at 155 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab137076 was shown to specifically react with BAT3/BAG-6 when BAT3/BAG-6 knockout samples were used. Wild-type and BAT3/BAG-6 knockout samples were subjected to SDS-PAGE.  Ab137076 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.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-BAG6 knockout cells (red line) stained with ab137076. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1 x PBS/10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab137076, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-BAG6 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions. 

Immunohistochemical analysis of Paraffin-embedded human testis tissue sections labeling BAT3/BAG-6 with purified ab137076 at a dilution of 1/50 dilution (7.1 μg/ml). ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 was used as the secondary anitbody. Sections were counterstained with hematoxylin. Antigen retrieval was heat mediated using EDTA Buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling BAT3/BAG-6 with purified ab137076 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab195889 Anti-Alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200. DAPI (blue) was used as a nuclear counterstain. Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

Intracellular Flow Cytometry analysis of HeLa cells labelling BAT3/BAG-6 (red) with purified ab137076 at dilution of 1/30. Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 was used as the secondary antibody. Cells were fixed with 4% paraformaldehyde. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody. 

Overlay histogram showing Hela cells stained with ab137076 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab137076, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling BAT3/BAG-6 with unpurified ab137076 at 1/50 dilution.