All lanes : Anti-Glycogen synthase 1/GYS1 antibody [EP817Y] (ab40810) at 1/10000 dilution

Lane 1 : Wild-type HeLa lysate
Lane 2 : Glycogen synthase 1/GYS1 knockout HeLa lysate
Lane 3 : HEK-293 lysate
Lane 4 : Daudi lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 81 kDa

Lanes 1-4: Merged signal (red and green). Green - ab40810 observed at 80 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab40810 Recombinant Anti-Glycogen synthase 1/GYS1 antibody [EP817Y] was shown to specifically react with Glycogen synthase 1/GYS1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265388 (knockout cell lysate ab257462) was used. Wild-type and Glycogen synthase 1/GYS1 knockout samples were subjected to SDS-PAGE. ab40810 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 dilution and 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.

 

Anti-Glycogen synthase 1/GYS1 antibody [EP817Y] (ab40810) at 1/50000 dilution + HeLa cell lysate at 10 µg

Predicted band size: 81 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Overlay histogram showing HEK293 cells stained with ab40810 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40810, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HEK293 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab40810 at a 1:250 dilution staining HeLa cells using anti-Glycogen Synthase 1/GYS1 RabMAb.