Anti-IRF3 antibody [EP2419Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EP2419Y] (AB76409)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling IRF3 with Purified 76409 at 1/20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EP2419Y] (AB76409)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue sections labeling IRF3 with Purified 76409 at 1 : 100 dilution (1.55 µg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Lab

Immunoprecipitation - Anti-IRF3 antibody [EP2419Y] (AB76409)

Purified ab76409 at 1/20 dilution (0.8μg) immunoprecipitating IRF3 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab76409 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab76409 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 47 kDa
Fresh lysate need to be used to avoid protein degradation.

All lanes:

Immunoprecipitation - Anti-IRF3 antibody [EP2419Y] (ab76409)

Predicted band size: 47 kDa

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• WB

Unknown

Western blot - Anti-IRF3 antibody [EP2419Y] (AB76409)

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-IRF3 antibody [EP2419Y] (ab76409) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 3:

U-937 (Human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 4:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 5:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 47 kDa

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• WB

Lab

Western blot - Anti-IRF3 antibody [EP2419Y] (AB76409)

Lanes 1 - 2 : Merged signal (red and green). Green - ab76409 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab76409 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 ab76409 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:

Western blot - Anti-IRF3 antibody [EP2419Y] (ab76409) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

IRF3 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human IRF3 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-irf3-knockout-a549-cell-line-ab267097'>ab267097</a>)

Predicted band size: 47 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-IRF3 antibody [EP2419Y] (AB76409)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76409 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab76409 was shown to react with IRF3 in wild-type HeLa. 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. ab76409 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.

All lanes:

Western blot - Anti-IRF3 antibody [EP2419Y] (ab76409) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human IRF3 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-irf3-knockout-hela-cell-line-ab255345'>ab255345</a>)

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

IRF3 knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 47 kDa

Observed band size: 47 kDa,50 kDa

false

• WB

Unknown

Western blot - Anti-IRF3 antibody [EP2419Y] (AB76409)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76409 observed at 50 kDa. Red - loading control, ab8245, observed at 37kDa.

ab76409 was shown to react with IRF3 in wild-type HAP1 cells alond with additional cross-reactive bands. No band was observed when IRF3 knockout samples were used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab76409 and ab8245 (loading control to GAPDH) were both diluted 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.

All lanes:

Western blot - Anti-IRF3 antibody [EP2419Y] (ab76409) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

IRF3 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 47 kDa

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