All lanes : Anti-HDAC6 antibody [EPR1698(2)] (ab133493) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HDAC6 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 131 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab133493 observed at 160 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab133493 was shown to react with HDAC6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264804 (knockout cell lysate ab257145) was used. Wild-type HeLa and HDAC6 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. ab133493 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: 5 bp deletion in exon 3.

 

Allele-2: 3 bp deletion in exon 3.

 

Representative images of HDAC6 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.