All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/500 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : IRF5 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 58 kDa

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).

Immunocytochemistry/ Immunofluorescence analysis of IRF5 in Raw264.7 cells. The cell was stained with ab33478 (1:100). The secondary antibody (green) was used Alexa Fluor 488. DAPI was stained the cell nucleus (blue).

Immunocytochemistry/ Immunofluorescence analysis of IRF5 in THP-1 cells. The cell was stained with ab33478 (1:100). The secondary antibody (green) was used Alexa Fluor 488. DAPI was stained the cell nucleus (blue).

Anti-IRF5 antibody [10T1] (ab33478) at 1/1000 dilution + THP-1 cell lysate

Secondary
Goat anti-mouse secondary antibody conjugated to HRP

Predicted band size: 58 kDa

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. 

All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/1000 dilution

Lane 1 : Ramos cell lysate
Lane 2 : THP-1 cell lysate
Lane 3 : A20 cell lysate
Lane 4 : NIH 3T3 cell lysate

Secondary
All lanes : goat anti-mouse secondary antibody conjugated to HRP

Developed using the ECL technique.

Predicted band size: 58 kDa
Observed band size: 58 kDa
Additional bands at: 26 kDa, 80 kDa. We are unsure as to the identity of these extra bands.

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.