BCAR4 Antibody / Breast cancer anti-estrogen resistance protein 4 (FY13054)

Flow Cytometry analysis of 293T cells using anti-BCAR4 antibody. Overlay histogram showing 293T cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-BCAR4 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of BCAR4 using anti-BCAR4 antibody. Lane 1: rat C6 whole cell lysates, Lane 2: rat PC-12 whole cell lysates, Lane 3: rat RH35 whole cell lysates, Lane 4:mouse RAW265.7 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-BCAR4 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. A single band is detected at approximately 16 kDa, slightly above the predicted molecular weight of 13 kDa. The higher apparent size is consistent with the anomalous migration of small, hydrophobic membrane-associated proteins on SDS-PAGE due to limited SDS binding.