All lanes : Anti-CaMKII delta antibody [EPR13095] (ab181052) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CAMK2D knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 56 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab181052 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab181052 was shown to react with CaM-kinase II  in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267322 (knockout cell lysate ab257376) was used. Wild-type HEK-293T and CAMK2D knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab181052 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: 14 bp deletion in exon5

 

Allele-2: 1 bp insertion in exon 5.

 

Representative images of CAMK2D knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.