Recombinant 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.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Galactosidase with purified ab168341 at 1/500. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

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.

Blocking and dilution buffer: 5% NFDM/TBST.

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.

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.

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. 

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.

Blocking and dilution buffer: 5% NFDM/TBST.

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.