Anti-IRF5抗体[10T1] (ab33478)

Western blot: Anti-IRF5 antibody [10T1] (ab33478) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab33478 was shown to bind specifically to IRF5. A band was observed at 63 kDa in wild-type A549 cell lysates with no signal observed at this size in IRF5 knockout cell line. To generate this image, wild-type and IRF5 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

Lanes 1 - 2: Merged signal (red and green). Green - ab33478 observed at 56 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab33478 was shown to specifically react with IRF5 in wild-type HAP1 cells as signal was lost in IRF5 knockout cells. Wild-type and IRF5 knockout samples were subjected to SDS-PAGE. Ab33478 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution 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.

This image was produced using the ascites version of this antibody. 

Flow cytometry analysis of IRF5 in THP-1 cell line, staining at 2-5µg for 1x10⁶ cells (red line). The secondary antibody used goat anti-mouse IgG Alexa fluor 488 conjugate. Isotype control antibody was mouse IgG (black line).

Immunofluorescence/Immunocytochemistry analysis of HEK293 cells (10⁵-10⁶) stained with Mouse monoclonal [10T1] to IRF5 (ab33478 1/100-1/200).

This image was produced using the ascites version of this antibody. 

100-200µg of whole cell lysates were used for western blotting.

This image was produced using the ascites version of this antibody. 

Overlay histogram showing THP1 cells stained with ab33478 (red line). The cells were fixed with 4% paraformaldehyde (10 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. The cells were then incubated with the antibody (ab33478, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in THP1 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

This image was produced using the ascites version of this antibody.