The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. Detects a band of approximately 25 kDa (predicted molecular weight: 23 kDa).
In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti-apoptotic action of BCL2 or its adenovirus homolog E1B 19k protein. Low micromolar levels of zinc ions inhibit the promotion of apoptosis.
Expressed in a wide variety of tissues, with highest levels in the heart and skeletal muscle.
Belongs to the Bcl-2 family.
Intact BH3 motif is required by BIK, BID, BAK, BAD and BAX for their pro-apoptotic activity and for their interaction with anti-apoptotic members of the Bcl-2 family.
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: BAK knockout HAP1 whole cell lysate (20 µg)
Lanes 1 - 2: Merged signal (red and green). Green - ab92999 observed at 23 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab92999 was shown to recognize BAK in wild-type HAP1 cells as signal was lost at the expected MW in BAK knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and BAK knockout samples were subjected to SDS-PAGE. Ab92999 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 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.
Western blot - Bak antibody (ab92999)
Anti-Bak antibody (ab92999) at 1 µg/ml + A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Immunocytochemistry/ Immunofluorescence - Bak antibody (ab92999)
ICC/IF image of ab92999 stained HepG2 cells. The cells were 4% formaldehyde fixed (5 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 ab92999 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit IgG (H+L)(ab96899) used at a 1/1000 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.