重组Anti-p40-phox抗体[EP2142Y] (ab76158)

ICC/IF image of ab76158 stained MCF7 cells. The cells were 4% formaldehyde 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 (ab76158, 1/25 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

ab76158 at 1/100-1/250 dilution staining p40-phox in human spleen by Immunohistochemistry, Paraffin-embedded tissue.

Overlay histogram showing K562 cells stained with ab76158 (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 (ab76158, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

p40-phox was immunoprecipitated from 0.35 mg HL-60 (Human acute promyelocytic leukemia promyeloblast) whole cell lysate 10 µg with 76158 at 1/50 dilution (2µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HL-60 (Human acute promyelocytic leukemia promyeloblast) whole cell lysate 10 µg

Lane 2: ab76158 IP in HL-60 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab76158 in HL-60 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.