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Synthetic peptide corresponding to Bcl-2 aa 41-54.
Our Abpromise guarantee covers the use of ab692 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 5 µg/ml.|
ab692 staining Bcl-2 in SK-N-SH cells treated with (R)-(-)-Deprenyl hydrochloride (Selegiline hydrochloride) (ab120604), by ICC/IF. Increase of Bcl-2 expression correlates with increased concentration of (R)-(-)-Deprenyl hydrochloride (Selegiline hydrochloride), as described in literature.
The cells were incubated at 37°C for 3h in media containing different concentrations of ab120604 ((R)-(-)-Deprenyl hydrochloride (Selegiline hydrochloride)) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab692 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A anti-mouse DyLight 488 polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
ab692 diluted 1/100 in BSA was incubated with whole cell lysate of human lymphocytes and a Protein G matrix for 12 hours at 4°C to achieve immunoprecipitation. 1,000,000 cells were present in the lysate.
A modified RIPA buffer was used to lyse cells.
The IP was washed with PBS/Triton.
ab692 was used at a 1/1000 dilution for the Western Blot step.
Bcl-2 was localized distinctly in specific cytoplasmic regions in human epithelium (especially in the lower and outmost layers). This image was kindly supplied as part of the review submitted by Marko Nykanen.
Overlay histogram showing Jurkat cells stained with ab692 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab692, 1/10 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
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