Lanes 1 - 4: Merged signal (red and green). Green - ab18256 observed at 29 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab18256 was shown to recognize HMGB1 in wild-type HAP1 cells as signal was lost at the expected MW in HMGB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. Ab18256 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.

ab18256 stained inHeLa cells. Cells were fixed with 4% paraformaldehyde (10min) 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 ab18256 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilutionovernight at +4°C. The secondary antibodies wereGoat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120)(pseudo-colored red)andGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody(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

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