Allele-1: 5 bp deletion in exon2

 

All lanes : Anti-Synaptophysin antibody [YE269] (ab32127) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32127 observed at 38 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32127 was shown to react with Syp in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255356 (knockout cell lysate ab263862) was used. Wild-type HEK-293T and SYP knockout HEK-293T 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. ab32127 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-2: 1 bp deletion in exon 2.