Anti-Bid antibody [Y8]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Bid antibody [Y8] (AB32060)

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 >5000 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bid antibody [Y8] (AB32060)

Immunocytochemistry analysis of MCF7 (human breast adenocarcinoma epithelial cell) labeling Bid with purified ab32060 at 1/500 dilution (10 µg/ml). Cells were fixed with 100% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control : PBS instead of the primary antibody.

• IP

Lab

Immunoprecipitation - Anti-Bid antibody [Y8] (AB32060)

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

All lanes:

Immunoprecipitation - Anti-Bid antibody [Y8] (ab32060)

Predicted band size: 22 kDa

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• WB

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Western blot - Anti-Bid antibody [Y8] (AB32060)

All lanes:

Western blot - Anti-Bid antibody [Y8] (ab32060) at 1/1000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

• WB

AbReview24935****

Western blot - Anti-Bid antibody [Y8] (AB32060)

All lanes:

Western blot - Anti-Bid antibody [Y8] (ab32060) at 1/500 dilution

Lanes 1 - 2:

Whole cell lysate prepared from breast cancer clinical extract at 20 µg

Lane 3:

Whole cell lysate prepared from MCF7 cells at 20 µg

Secondary

All lanes:

Goat anti-rabbit IgG-HRP at 1/1000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

true

Exposure time: 25min

Image courtesy of an anonymous Abreview.