All lanes : Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : USP9X knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 292 kDa
Observed band size: 290 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab180191 observed at 290 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab180191 was shown to react with USP9x in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265665 (knockout cell lysate ab257790) was used. Wild-type HeLa and USP9X knockout HeLa 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. ab180191 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated 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 exon 9.

 

All lanes : Anti-USP9x antibody (ab19879) at 1 µg/ml

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : USP9X knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 292 kDa
Observed band size: 290 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab19879 observed at 290 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab19879 was shown to react with USP9x in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265665 (knockout cell lysate ab257790) was used. Wild-type HeLa and USP9X knockout HeLa 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. ab19879 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 µg/ml 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-2: 1 bp insertion in exon 9.