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Apoptosis is related to many diseases and development. The p53 tumor-suppressor protein induces apoptosis through transcriptional activation of several genes. A novel p53 inducible pro-apoptotic gene was identified recently and designated PUMA (for p53 upregulated modulator of apoptosis) and bbc3 (for Bcl-2 binding component 3) in human and mouse (1-3). PUMA/bbc3 is one of the pro-apoptotic Bcl-2 family members including Bax and Noxa, which are also transcriptional targets of p53. The PUMA gene encodes two BH3 domain-containing proteins termed PUMA-a and PUMA-b (1). PUMA proteins bind Bcl-2, localize to the mitochondria, and induce cytochrome c release and apoptosis in response to p53. PUMA may be a direct mediator of p53-induced apoptosis.
Our Abpromise guarantee covers the use of ab9643 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 23 kDa.Can be blocked with Human PUMA peptide (ab9644). Use at a concentration of 1 - 2 µg/ml. Detects a band of approximately 23 kDa. Can be blocked with PUMA peptide (180/193) (ab9644). A lower band at approximately 16 kDa was detected in MOLT4 and U937 cells, which may represent the PUMA-beta form.|
|IHC-P||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 µg/ml.|
Blocked with 5% milk for 1 hour.
Incubated with the primary antibody for 18 hours.
ab9643 staining rat gonad tissue sections by Immunohistochemistry (IHC-P - Formalin/PFA-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 1.5% milk for 45 minutes at 25°C. Protease/PBS (100mg/200ml) was used for antigen retrieval, H2O2/methanol solution (0.3%) was used to destroy endogenous peroxidase activity. Samples were incubated with the primary antibody (1/500) for 12 hours at 4°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.
ICC/IF image of ab9643 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9643, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
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