Anti-BNIP3抗体[ANa40] (ab10433)

Flow cytometry 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-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab10433) (1x10⁶ in 100μl at 5.0μg/ml) for 30 min at 22°C.

The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150117) preadsorbed was incubated at 1/4000 for 30 min at 22°C.

Isotype control antibody (black line) was Mouse IgG2b, kappa monoclonal [7E10G10] (ab170192) used under the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

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.

Immunohistochemical analysis of Human laryngeal squamous cell carcinoma tissue, staining BNIP3 with ab10433.

Antigen retrieval was performed by heat mediation in citrate buffer. The sections were incubated with primary antibody (1/100) overnight at 4°C in a humidified chamber. Staining was visualized using DAB, followed by hematoxylin nuclear counterstaining.

Anti-BNIP3 antibody [ANa40] (ab10433) at 1 µg/ml + Human skeletal muscle tissue lysate - total protein (ab29330) at 20 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 30 kDa


Exposure time: 5 seconds

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 BNIP3 antibody staining in a section of formalin-fixed paraffin-embedded normal human kidney* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab10433, 1ug/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre