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

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.

Allele-1: 1 bp deletion in exon7

 

Allele-2: 1 bp insertion in exon 7.