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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung cancer tissue sections labeling GBA with Purified ab125065 at 1:50 dilution (2.1 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : GBA knockout HAP1 whole cell lysate
Lane 3 : MCF7 whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 60 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab125065 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab125065 was shown to specifically recognize GBA in wild-type HAP1 cells as well as additional cross-reactive bands. No bands were observed when GBA knockout samples were used. Wild-type and GBA knockout samples were subjected to SDS-PAGE. Ab125065 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

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

Lane 1 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysates
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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

The multiple bands are consistent with what have been described in the literature PMID: 2495719 due to the glycosylation

ab125065, at a 1/50 dilution, staining GBA in paraffin-embedded Human colon tissue by immunohistochemistry. Heat mediated antigen retrieval was permorded using citrate buffer pH 6 before commencing with IHC staining protocol.

This image was generated using the unpurified version of the product. 

 

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

Lane 1 : Saos-2 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : SH-SY5Y cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 60 kDa

This image was generated using the unpurified version of the product.