All lanes : Anti-GRB2 antibody [Y301] (ab32111) at 1/2000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : GRB2 knockout HCT116 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 25 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32111 observed at 25 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32111 was shown to react with GRB2 in wild-type HCT 116 cells in western blot with loss of signal observed in GRB2 knockout cell line ab273715 (GRB2 knockout cell lysate ab275248). Wild-type and GRB2 knockout HCT 116 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab32111 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 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.

Allele-1: 2 bp deletion in exon 2 .

Allele-2: 1 bp deletion in exon 2 .