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

This data was developed using the same antibody clone in a different buffer formulation (ab92324).

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 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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?

This data was developed using the same antibody clone in a different buffer formulation (ab92324).

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?

This data was developed using ab92324, the same antibody clone in a different buffer formulation.

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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

ICC/IF image of unpurified ab92324 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab92324, neat) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92324).

This IHC data was generated using the same anti-TNFAIP3 antibody clone, EPR2663, in a different buffer formulation (cat# ab92324).

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.