All lanes : Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 60 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab128879 observed at 60 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab128879 was shown to react with GBA in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265038 (knockout cell lysate ab256929) was used. Wild-type HeLa and GBA 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. ab128879 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.

All lanes : Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab125065 observed at 70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab125065 was shown to react with GBA in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265038 (knockout cell lysate ab256929) was used. Wild-type HeLa and GBA 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. ab125065 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.

Homozygous: 1 bp insertion in exon 3.

Representative images of GBA 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.