Recombinant Anti-IRF3 antibody [EPR2418Y] (ab68481)

Lanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab68481 was shown to react with IRF3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255345 (knockout cell lysate ab263784) was used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab68481 was shown to react with IRF3 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when IRF3 knockout samples were examined. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and ab8245 (loading control to GAPDH) were both diluted to 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-IRF3 knockout cells (red line) stained with ab68481. The cells were fixed with 80% methanol (5 min) (left pannel) or 4% formaldehyde (10 min) (right pannel), and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab68481, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-IRF3  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

Note: We recommend fixing cells using MeOH instead of PFA to get optimal results.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling IRF3 with ab68481 at 1/100 dilution. Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/400 dilution was used as the secondary antibody (green). The confocal image shows cytoplasmic on HeLa cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab68481 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

Blocking and Dilution buffer: 5% NFDM/TBST

Blocking and Dilution buffer: 5% NFDM/TBST

Blocking and Dilution buffer: 5% NFDM/TBST

Blocking and Dilution buffer: 5% NFDM/TBST

The slightly smaller molecular mass observed in mouse than in human is supported by literature.

Flow cytometry analysis of 2% paraformaldehyde fixed U937 (Human histiocytic lymphoma cells) cells labeling IRF3 with ab68481 at 1/160 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

Immunohistochemical analysis of paraffin-embedded Human tonsil labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse spleen labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.