Anti-IRF3 抗体 [EPR2418Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (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 MeOHinstead of PFA toget optimal results.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Intracellular 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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-IRF3 antibody [EPR2418Y] (AB68481)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

• WB

Lab

Western blot - 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 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.

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) 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

false

• WB

Lab

Western blot - Anti-IRF3 antibody [EPR2418Y] (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.

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) 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='/products/cell-lines/human-irf3-knockout-a549-cell-line-ab267097'>ab267097</a>)

Predicted band size: 47 kDa

Observed band size: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab320082 was shown to bind specifically to IRF3. Target of interest was observed at 60 kDa in wild-type Hela cell lysates (lane 2) with no signal observed at this size in IRF3 knockout cell line (lane 4) (lane 4, knockout cell line ab255345 / knockout cell lysate ab263784).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-IRF3 antibody [EPR2418Y] (ab68481) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-IRF3 (phospho S396) antibody [EPR28686-189] (<a href='/products/primary-antibodies/irf3-phospho-s396-antibody-epr28686-189-ab320082'>ab320082</a>) at 1/1000 dilution

Lane 1:

Untreated wild-typeHeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 60 µg

Lane 2:

Wild-type HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (untreated membrane) at 60 µg

Lane 3:

Untreated IRF3 knockout HeLa whole cell lysate (untreated membrane) at 60 µg

Lane 4:

IRF3 knockout HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (untreated membrane) at 60 µg

Lane 5:

Untreated wild-typeHeLa whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

Lane 6:

Wild-type HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

Lane 7:

Untreated IRF3 knockout HeLa whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

Lane 8:

IRF3 knockout HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 60 kDa,36 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The identity of the bands between 25 kDa and 35 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-IRF3 antibody [EPR2418Y] (ab68481) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-IRF3 (phospho S396) antibody [EPR28686-189] (<a href='/products/primary-antibodies/irf3-phospho-s396-antibody-epr28686-189-ab320082'>ab320082</a>) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 60 µg

Lane 2:

THP-1 treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate at 60 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 60 kDa,36 kDa

false

Exposure time: 48s

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/10000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 20 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 51 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) 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='/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='/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: 37 kDa,51 kDa

false

• WB

Lab

Western blot - Anti-IRF3 antibody [EPR2418Y] (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:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) 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='/products/cell-lines/human-irf3-knockout-a549-cell-line-ab267098'>ab267098</a>)

Predicted band size: 47 kDa

Observed band size: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/10000 dilution

Lane 1:

THP-1 (Human monocytic leukemia cells) whole cell lysates at 10 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysates at 10 µg

Lane 3:

Daudi (Human Burkitt's lymphoma cell line) whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 51 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481)

Blocking and Dilution buffer : 5% NFDM/TBST

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

All lanes:

Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse spleen lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 47 kDa

false