All lanes : Anti-HP1 gamma/CBX3 antibody [EPR19802] (ab217999) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CBX3 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab217999 observed at 25 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab217999 was shown to react with HP1 gamma/CBX3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261744 (knockout cell lysate ab257110) was used. Wild-type HeLa and CBX3 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. ab217999 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 2000 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: 5 bp deletion in exon 2.

 

Allele-2: 4 bp deletion in exon 2.