Lanes 1 - 4: Merged signal (red and green). Green - ab47062 observed at 150 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab47062 was shown to recognize TRIM33 in wild-type HAP1 cells as signal was lost at the expected MW in TRIM33 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TRIM33 knockout samples were subjected to SDS-PAGE. Ab47062 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

The 150 kDa band observed is comparable to molecular weights seen with other commercially available antibodies to TRIM33. 

ab47062 stained in MCF7 cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab47062 at 5 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.