Astana Biomed Group, an authorized Abcam distributor in Central Asia

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

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP5828(2)] to Galactosidase alpha
  • Suitable for: Flow Cyt, IP, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-Galactosidase alpha antibody [EP5828(2)]
    See all Galactosidase alpha primary antibodies

  • Description

    Rabbit monoclonal [EP5828(2)] to Galactosidase alpha

  • Host species

    Rabbit

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide within Human Galactosidase alpha aa 100-200. The exact sequence is proprietary.
    Database link: P06280

  • Positive control

    • WB: MCF-7, 293T, A431, HAP1 and HeLa whole cell lysate (ab150035). IHC-P: Human urinary bladder carcinoma, kidney and uterus tissue. ICC/IF: HeLa cells IP: MCF-7 cell lysates. Flow Cyt: HeLa cells.

  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.

  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol, 0.05% BSA, 59% PBS
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EP5828(2)

  • Isotype

    IgG

  • Research areas

Images

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

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : GLA knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 49 kDa
    Observed band size: 49 kDa



    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 - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Immunocytochemistry/ Immunofluorescence - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)

    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).

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

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : GLA knockout HAP1 whole cell lysate
    Lane 3 : Hela whole cell lysate
    Lane 4 : MCF-7 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 49 kDa



    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.

  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 20 µg (purified) + MCF-7 whole cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 49 kDa
    Observed band size: 46 kDa
    why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    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.

  • Flow Cytometry - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Flow Cytometry - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)

    Flow Cytometry analysis of HeLa 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.

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

  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/5000 dilution (purified) + HEK293 whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 49 kDa
    Observed band size: 46 kDa why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    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.

  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
    All lanes : Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341) at 1/1000 dilution (unpurified)

    Lane 1 : MCF-7 cell lysates
    Lane 2 : 293T cell lysates
    Lane 3 : A431 cell lysates
    Lane 4 : HeLa cell lysates

    Lysates/proteins at 10 µg per lane.

    Predicted band size: 49 kDa

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

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