Anti-GBA antibody [EPR5142]

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-GBA antibody [EPR5142] (AB125065)

IHC image of GBA staining in a section of frozen normal human placenta* performed on a Leica BOND™ 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-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5142] (AB125065)

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.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-GBA antibody [EPR5142] (AB125065)

IHC image of GBA staining in a section of frozen normal human hippocampus* performed on a Leica BOND™ 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-GBA antibody [EPR5142] (AB125065)

Negative control image : IHC image of GBA staining in a section of frozen normal human heart* performed on a Leica BOND™ 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-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5142] (AB125065)

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.

• WB

Lab

Western blot - Anti-GBA antibody [EPR5142] (AB125065)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of MCF7 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab125065 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab125065 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

All lanes:

MCF7 whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-GBA antibody [EPR5142] (AB125065)

ab125065 was shown to react with GBA1 in wild-type HAP1 cells in Western blot with loss of signal observed in a GBA1 knockout cell line. Wild-type HAP1 and GBA1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab125065 overnight at 4 °C at a 1/100 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/100 dilution

Lane 1:

wild-type HAP1 cells

Lane 2:

GBA1 knockout cell line

false

• WB

Lab

Western blot - Anti-GBA antibody [EPR5142] (AB125065)

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

All lanes:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

Lane 1:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 60 kDa

Observed band size: 60-70 kDa

false

• WB

Lab

Western blot - Anti-GBA antibody [EPR5142] (AB125065)

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:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

GBA knockout HAP1 whole cell lysate at 40 µg

Lane 3:

MCF7 whole cell lysate at 40 µg

Lane 4:

HepG2 whole cell lysate at 40 µg

Predicted band size: 60 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

GBA knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human GBA knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-gba-knockout-hela-cell-line-ab265038'>ab265038</a>)

Predicted band size: 60 kDa

Observed band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-GBA antibody [EPR5142] (AB125065)

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

All lanes:

Western blot - Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

Lane 1:

Saos-2 cell lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

SH-SY5Y cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 60 kDa

true