All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : TNFAIP3 knockout A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : TNFAIP3 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 90 kDa
Observed band size: 90 kDa

Lanes 1- 4: Merged signal (red and green). Green - ab92324 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab92324 was shown to react with TNFAIP3 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab266946 (knockout cell lysate ab257114) was used. Wild-type A549 and TNFAIP3 knockout A549 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab92324 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling TNFAIP3 with unpurified ab92324 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. Counterstained with Hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labelling TNFAIP with purified ab92324 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TNFAIP3 knockout HeLa cell lysate
Lane 3 : Jurkat cell treated with 5ng/ml PMA for 48 hours and then treated with 2µg/ml PHA for 48 hours, whole cell lysate
Lane 4 : Untreated Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 90 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab92324 observed at 80 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab92324 Anti-TNFAIP3 antibody [EPR2663] was shown to specifically react with TNFAIP3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265983 (knockout cell lysate ab257112) was used. Wild-type and TNFAIP3 knockout samples were subjected to SDS-PAGE. ab92324 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 10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/1000 dilution

Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : TNFAIP3 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) cell treated with 5ng/ml PMA for 48 hours and then treated with 2µg/ml PHA for 48 hours, whole cell lysate
Lane 4 : Untreated Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 90 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab92324 observed at 80 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab92324 Anti-TNFAIP3 antibody [EPR2663] was shown to specifically react with TNFAIP3 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266945 (knockout cell lysate ab257113) was used. Wild-type and TNFAIP3 knockout samples were subjected to SDS-PAGE. ab92324 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 : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/5000 dilution

Lane 1 : WEHI-3 (Mouse leukemia lymphoblast) whole cell lysate
Lane 2 : WEHI-3 treated with 20 ng/ml TNF alpha (ab9642) for 6 h

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 90 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/2000 dilution (unpurified) + Jurkat cell lysate - treated with TNF (ab9642) and TPA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 90 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/3000 dilution (purified) + Jurkat cell lysate - treated with TNF (ab9642) and TPA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 90 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling TNFAIP3 with purified ab92324 at 1/100. 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. Counterstained with Hematoxylin.

Immunocytochemsitry/Immunofluorescence analysis of Daudi cells labelling TNFAIP3 (red) with unpurified ab92324 at 1/50. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

Immunocytochemsitry/Immunofluorescence analysis of Daudi cells labelling TNFAIP3 (red) with purified ab92324 at 1/100. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

Overlay histogram showing HepG2 cells stained with unpurified ab92324 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab92324, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Flow cytometry analysis of Daudi cells labelling TNFAIP3 with unpurified ab92324 at 1/20 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

Flow cytometry analysis of Daudi cells labelling TNFAIP3 with purified ab92324 at 1/30 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/1000 dilution (unpurified)

Lane 1 : Jurkat cells treated with TNF (ab9642) and TPA
Lane 2 : Daudi cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 90 kDa