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Recombinant fragment (Residues 1-163 of Human BNIP3).
Our Abpromise guarantee covers the use of ab10433 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 21.5 kDa).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|Flow Cyt||Use 0.1-1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use at an assay dependent concentration.|
IHC image of ab10433 staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10433, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab10433 staining BNIP3 in Human fibroblasts by Immunocytochemistry/ Immunofluorescence. Cells were fixed in 2% paraformaldehyde for 30 minutes at room temperature and cells were treated with 50 mM NH4Cl in PBS for 10 minutes to quench free aldehyde groups after permeabilisation. Cells were then permeabilized in 0.1% Triton X-100 prior to blocking in 1% BSA for 1 hour at room temperature. The primary antibody was diluted 1/400 and incubated with the sample for 1 hour at room temperature. The secondary antibody was Alexa Fluor® 555-conjugated goat anti-mouse polyclonal, diluted 1/1000.
Overlay histogram showing HepG2 cells stained with ab10433 (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 (ab10433, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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