Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: IRF2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: CACO2 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab124744 observed at 48 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab124744 was shown to recognize IRF2 when IRF2 knockout samples were used, along with additional cross-reactive bands. Wild-type and IRF2 knockout samples were subjected to SDS-PAGE.  Ab124744 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 and 1/10000 dilutions respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) ab216773 and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IRF2 antibody [EPR4644(2)] (ab124744) at 1/5000 dilution

Lane 1 : SW480 (Human colorectal adenocarcinoma cell line)
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma)
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood)

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 39 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon tissue labeling IRF2 with purified ab124744 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution was used as the secondary antibody. Nucleus staining on epithelium of human colon was observed. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling IRF2 with ab124744 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab124744 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

All lanes : Anti-IRF2 antibody [EPR4644(2)] (ab124744) at 1/1000 dilution

Lane 1 : Caco-2 (Human colorectal adenocarcinoma cells)
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus)
Lane 3 : NIH/3T3 (Mouse embyro fibroblast cells)

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 39 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical cancer tissue labeling IRF2 with purified ab124744 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution was used as the secondary antibody. Nucleus staining on tumor cells of human cervix cancer was observed. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

Anti-IRF2 antibody [EPR4644(2)] (ab124744) at 1/1000 dilution + Human fetal lung lysate at 10 µg

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

Predicted band size: 39 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse colon tissue labeling IRF2 with purified ab124744 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution was used as the secondary antibody. Nucleus staining on epithelial cells of mouse colon was observed. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic carcinoma tissue labelling IRF2 with purified ab124744 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

Equilibrium disassociation constant (K_D)
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