Recombinant Anti-Bid antibody [Y8] (ab32060)

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using ab32060 at 1/100 dilution.

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

Purified ab32060 at 1/50 dilution (2µg) immunoprecipitating Bid in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab32060 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32060 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 22 kDa

Overlay histogram showing HeLa cells stained with ab32060 (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 (ab32060, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). 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.

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