Recombinant Anti-GBA antibody [EPR5142] (ab125065)

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.

IHC image of GBA staining in a section of frozen normal human hippocampus* performed on a Leica BONDTM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab125065, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of GBA staining in a section of frozen normal human placenta* performed on a Leica BONDTM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab125065, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Negative control image: IHC image of GBA staining in a section of frozen normal human heart* performed on a Leica BONDTM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab125065, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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.

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. 

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