All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BRAF knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 84 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab33899 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab33899 was shown to react with BRAF in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265373 (knockout cell lysate ab257078) was used. Wild-type HeLa and BRAF knockout HeLa 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. ab33899 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: 10 bp deletion in exon 3.

 

Allele-2: 1 bp insertion in exon 3.