Anti-Galactosidase alpha antibody [EP5828(2)]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Intracellular Flow Cytometry analysis ofHeLa cells labelling Galactosidase alpha with purified ab168341 at a dilution of 1/90 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Flour^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human urinary bladder carcinoma tissue labelling Galactosidase alpha with unpurified ab168341 at a dilution of 1/50.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human uterus tissue labelling Galactosidase alpha with unpurified ab168341 at a dilution of 1/50.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling Galactosidase alpha with purified ab168341 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Lab

Immunoprecipitation - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

ab168341 (purified) at 1/60 immunoprecipitating Galactosidase alpha in MCF-7 whole cell lysate.

Lane 1 (input) : MCF-7 whole cell lysate (10μg)

Lane 2 (+) : ab168341 + MCF-7 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab168341 in MCF-7 whole cell lysate.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)

Predicted band size: 49 kDa

Observed band size: 46 kDa

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• WB

Lab

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Lanes 1-2 : Merged signal (red and green). Green - ab168341 observed at 49 kDa. Red - loading control ab8245 observed at 37 kDa.

ab168341 Anti-Galactosidase alpha antibody [EP5828(2)] was shown to specifically react with Galactosidase alpha in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265563 (knockout cell lysate ab257449) was used. Wild-type and Galactosidase alpha knockout samples were subjected to SDS-PAGE. ab168341 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 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-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

GLA knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human GLA (Galactosidase alpha) knockout HeLa cell line (<a href='/ko/products/cell-lines/human-gla-galactosidase-alpha-knockout-hela-cell-line-ab265563'>ab265563</a>)

Predicted band size: 38 kDa,49 kDa

Observed band size: 49 kDa

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• WB

Lab

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/5000 dilution

All lanes:

HEK293 whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 49 kDa

Observed band size: 46 kDa

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• WB

Lab

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Lanes 1 - 4 : Merged signal (red and green). Green - ab168341 observed at 49 kDa. Red - loading control, ab130007, observed at 125 kDa.

ab168341 was shown to recognize GLA (Alpha-galactosidase A) in wild-type HAP1 cells as signal was lost at the expected MW in GLA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GLA knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. ab168341 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GLA knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Hela whole cell lysate at 20 µg

Lane 4:

MCF-7 whole cell lysate at 20 µg

Predicted band size: 49 kDa

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• WB

Lab

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 20 µg

All lanes:

MCF-7 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 49 kDa

Observed band size: 46 kDa

false

• WB

Unknown

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (AB168341)

All lanes:

Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/1000 dilution

Lane 1:

MCF-7 cell lysates at 10 µg

Lane 2:

293T cell lysates at 10 µg

Lane 3:

A431 cell lysates at 10 µg

Lane 4:

HeLa cell lysates at 10 µg

Predicted band size: 49 kDa

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