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: 89 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 [59A426] (ab13597) at 1/500 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: 89 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 - ab13597 observed at 80 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab13597 Anti-TNFAIP3 antibody [59A426] 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. ab13597 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

Homozygous: 1 bp insertion in exon 7.

Representative images of TNFAIP3 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.