All lanes : Anti-FADD antibody [EPR4415] (ab108601) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FADD knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 23 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab108601 observed at 23 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108601 was shown to react with FADD in wild-type HeLa cells in western blot with loss of signal observed in FADD knockout cell line ab261817 (FADD knockout cell lysate ab257261). Wild-type and FADD knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108601 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-FADD antibody [OTI1C11] (ab119059) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FADD knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

Predicted band size: 23 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab119059 observed at 25 kDa. Red - loading control ab181602 observed at 37 kDa.

 ab119059 Anti-FADD antibody [OTI1C11] was shown to specifically react with FADD in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261817 (knockout cell lysate ab257261) was used. Wild-type and FADD knockout samples were subjected to SDS-PAGE. ab119059 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 2000 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 20000 dilution for 1 hour at room temperature before imaging.

 

Homozygous: Insertion of the selection cassette in exon 1.