Anti-BNIP3抗体[ANa40] (ab10433)

Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
Lane 2: BNIP3 knockout HAP1 whole cell lysate (40 µg)
Lane 3: SHSY5Y whole cell lysate (40 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab10433 observed at 30 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab10433 was shown to recognize BNIP3 in wild-type HAP1 cells as signal was lost at the expected MW in BNIP3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and BNIP3 knockout samples were subjected to SDS-PAGE. ab10433 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 µg/mL and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab10433 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406. 

IHC image of ab10433 staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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.

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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.