Anti-GBA antibody [EPR5143(3)]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] (AB128879)

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

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] (AB128879)

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

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GBA antibody [EPR5143(3)] (AB128879)

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.

• WB

Lab

Western blot - Anti-GBA antibody [EPR5143(3)] (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)] (ab128879) 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)] (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)] (ab128879) 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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• WB

Supplier Data

Western blot - Anti-GBA antibody [EPR5143(3)] (AB128879)

All lanes:

Western blot - Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/4000 dilution

Lane 1:

Saos-2 Cell Lysate at 20 µg

Lane 2:

U87-MG Cell Lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), HRP- conjugated at 1/1000 dilution

Predicted band size: 60 kDa

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• WB

Unknown

Western blot - Anti-GBA antibody [EPR5143(3)] (AB128879)

All lanes:

Western blot - Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

Saos-2 cell lysate at 10 µg

Lane 3:

U87-MG cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 60 kDa

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• WB

Unknown

Western blot - Anti-GBA antibody [EPR5143(3)] (AB128879)

Blocking and diluting buffers :

Lane 1 and 2 : 5% NFDM/TBST
Lane 3 and 4 : 2% BSA/TBST

We suggest using 2% BSA/TBST as blocking buffer and antibody diluting buffer if you cannot obtain strong band in some samples.

Lane 1:

Western blot - Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/10000 dilution

Lanes 2 - 4:

Western blot - Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/50000 dilution

All lanes:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/100000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

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Exposure time: 180s

• OI-RD Scanning

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OI-RD Scanning - Anti-GBA antibody [EPR5143(3)] (AB128879)

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.