All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : IRF3 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (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.

 

 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : IRF3 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab68481).

Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab68481 was shown to react with IRF3 in wild-type A549 cells in western blot with loss of signal observed in IRF3 knockout cell line ab267098 (IRF3 knockout cell lysate ab256954). Wild-type and IRF3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab68481 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : IRF3 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab68481).

Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab68481 was shown to react with IRF3 in wild-type A549 cells in western blot with loss of signal observed in IRF3 knockout cell line ab267097 (IRF3 knockout cell lysate ab256953). Wild-type and IRF3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab68481 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).