The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 26 kDa (predicted molecular weight: 26 kDa). Incubation 2hrs at room temperature
Use at 2 µg/mg of lysate.
Use a concentration of 2 - 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol, by boiling tissue sections in 10mM citrate buffer, pH 6.0.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
RelevanceBCL2 is an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes. Constitutive expression of BCL2, such as in the case of translocation of BCL2 to Ig heavy chain locus, is thought to be the cause of follicular lymphoma. Two transcript variants (alpha and beta) produced by alternate splicing, differ in their C-terminal ends.
BCL2 suppresses apoptosis in a variety of cell systems including factor-dependent lymphohematopoietic and neural cells. It regulates cell death by controlling the mitochondrial membrane permeability. It appears to function in a feedback loop system with caspases. BCL2 inhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and/or by binding to the apoptosis-activating factor (APAF1). It can form homodimers, and heterodimers with BAX, BAD, BAK and BclX(L). Heterodimerization with BAX requires intact BH1 and BH2 domains, and is necessary for anti-apoptotic activity. Also interacts with APAF1, RAF1, TP53BP2, BBC3, BCL2L1 and BNIPL.
Formalin-fixed, paraffin-embedded human tonsil with 2-4ug/ml ab77567, using peroxidase-conjugate and DAB chromogen. Note cytoplasmic and membrane staining of follicular cells and no staining of reactive cells in the germinal center.
Western blot - Bcl2 alpha antibody [100/D5] - Azide free (ab77567)
Anti-Bcl2 alpha antibody [100/D5] (ab77567) at 1 µg/ml + Human lymphoma cells lysate
Predicted band size : 26 kDa Observed band size : 26 kDa
Flow Cytometry-Anti-Bcl2 alpha antibody [100/D5] - BSA and Azide free(ab77567)
Overlay histogram showing HepG2 cells stained with ab77567 (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 (ab77567, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-Bcl2 alpha antibody [100/D5] (ab77567)
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