All lanes : Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab138501 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab138501 was shown to react with SNCA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255433 (knockout cell lysate ab263769) was used. Wild-type HEK-293T and SNCA 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. ab138501 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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: Insertion of the selection cassette in exon2

 

Allele-2: 1 bp insertion in exon 2.