Anti-GBA antibody [EPR5143(3)] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

Clone EPR5143(3) (ab215260) has been successfully conjugated by Abcam. This image was generated using Anti-GBA antibody [EPR5143(3)] (Alexa Fluor® 488). Please refer to ab225151 for protocol details.

IHC image of GBA staining in a section of formalin-fixed paraffin-embedded normal Human Kidney*. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20mins, performed on a Leica BOND^™. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab225151 at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

ab128879 staining GBA in Human kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/200). An undiluted HRP-conjugated anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128879).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

ab128879, unpurified, at a 1/100 dilution, staining GBA in paraffin embedded Human kidney tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128879).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

Clone EPR5143(3) (ab215260) has been successfully conjugated by Abcam. This image was generated using Anti-GBA antibody [EPR5143(3)] (Alexa Fluor® 647). Please refer to ab225150 for protocol details.

IHC image of GBA staining in a section of formalin-fixed paraffin-embedded normal Human Kidney*. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20mins, performed on a Leica BOND^™. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab225150 at 1/2500 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and 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 [EPR5143(3)] - BSA and Azide free (AB215260)

ab128879, unpurified, at a 1/100 dilution, staining GBA in paraffin embedded Human thyroid carcinoma tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128879).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

This data was developed using the same antibody clone in a different buffer formulation (ab128879).

Lanes 1 - 4 : Merged signal (red and green). Green - ab128879 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab128879 was shown to specifically react with 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. ab128879 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 800CW Goat anti-Rabbit and 680CW Goat anti-Mouse secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (ab215260) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GBA knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 60 kDa

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• WB

Lab

Western blot - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

This data was developed using the same antibody clone in a different buffer formulation (ab128879).

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:

Western blot - Anti-GBA antibody [EPR5143(3)] (<a href='/en-us/products/primary-antibodies/gba-antibody-epr51433-ab128879'>ab128879</a>) 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: 60 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (AB215260)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.