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    products/primary-antibodies/galactosidase-alpha-antibody-ep58282-ab168341.pdf

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Validated using a knockout cell lineRecombinantRabMAb

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

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Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Immunocytochemistry/ Immunofluorescence - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Western blot - 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) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Flow Cytometry (Intracellular) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Immunoprecipitation - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • Western blot - Anti-Galactosidase alpha antibody [EP5828(2)] (ab168341)
  • 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 (Intra), IP, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

You may also be interested in

Protein
Product image
Recombinant Human Galactosidase alpha protein (ab114549)
Primary
Product image
Anti-PAH antibody [EPR12380] (ab178430)
Knockout
Product image
Human GLA (Galactosidase alpha) knockout HeLa cell lysate (ab257449)

View more associated products

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

    Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • 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 (intra): HeLa cells.
  • General notes

    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.

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

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

    • Cardiovascular
    • Blood
    • Blood Cell Antigens
    • Cardiovascular
    • Blood
    • Other
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Anti-Galactosidase alpha antibody [EP5828(2)] - BSA and Azide free (ab215258)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human GLA (Galactosidase alpha) knockout HeLa cell line (ab265563)
  • KO cell lysates

    • Human GLA (Galactosidase alpha) knockout HeLa cell lysate (ab257449)
  • Positive Controls

    • HeLa whole cell lysate (ab150035)
    • MCF7 whole cell lysate (ab29537)
    • HeLa whole cell lysate (ab29545)
    • A-431 whole cell lysate (ab30132)
    • A-431 whole cell lysate (ab7909)
  • Recombinant Protein

    • Recombinant Human Galactosidase alpha protein (ab114549)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab168341 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
1/90.
IP
1/10 - 1/100.
WB (1)
1/1000 - 1/10000. Predicted molecular weight: 49 kDa.
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF
1/50 - 1/500.
Notes
Flow Cyt (Intra)
1/90.
IP
1/10 - 1/100.
WB
1/1000 - 1/10000. Predicted molecular weight: 49 kDa.
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF
1/50 - 1/500.

Target

  • Involvement in disease

    Defects in GLA are the cause of Fabry disease (FD) [MIM:301500]. FD is a rare X-linked sphingolipidosis disease where glycolipid accumulates in many tissues. The disease consists of an inborn error of glycosphingolipid catabolism. FD patients show systemic accumulation of globotriaoslyceramide (Gb3) and related glycosphingolipids in the plasma and cellular lysosomes throughout the body. Clinical recognition in males results from characteristic skin lesions (angiokeratomas) over the lower trunk. Patients may show ocular deposits, febrile episodes, and burning pain in the extremities. Death results from renal failure, cardiac or cerebral complications of hypertension or other vascular disease. Heterozygous females may exhibit the disorder in an attenuated form, they are more likely to show corneal opacities.
  • Sequence similarities

    Belongs to the glycosyl hydrolase 27 family.
  • Cellular localization

    Lysosome.
  • Target information above from: UniProt accession P06280 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 2717 Human
    • Omim: 300644 Human
    • SwissProt: P06280 Human
    • Unigene: 69089 Human
    • Alternative names

      • AGAL_HUMAN antibody
      • Agalsidase alfa antibody
      • Alpha D galactosidase A antibody
      • Alpha D galactoside galactohydrolase 1 antibody
      • Alpha D galactoside galactohydrolase antibody
      • Alpha gal A antibody
      • Alpha galactosidase A antibody
      • Alpha-D-galactosidase A antibody
      • Alpha-D-galactoside galactohydrolase antibody
      • Alpha-galactosidase A antibody
      • GALA antibody
      • Galactosidase, alpha antibody
      • GLA antibody
      • GLA protein antibody
      • Melibiase antibody
      see all

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

       

       

    • 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)

    Protocols

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (6)

    Publishing research using ab168341? Please let us know so that we can cite the reference in this datasheet.

    ab168341 has been referenced in 6 publications.

    • Lenders M  et al. Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease. Int J Mol Sci 22:N/A (2021). PubMed: 33800950
    • Zhu X  et al. Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates. Am J Hum Genet 104:625-637 (2019). PubMed: 30879639
    • Lenders M  et al. Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy. J Med Genet 56:548-556 (2019). PubMed: 31010832
    • Birket MJ  et al. A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology. Stem Cell Reports 13:380-393 (2019). PubMed: 31378672
    • Lenders M  et al. Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease. J Am Soc Nephrol 29:2879-2889 (2018). PubMed: 30385651
    • Fu C  et al. Puerarin protects endothelial progenitor cells from damage of angiotensin II via activation of ERK1/2-Nrf2 signaling pathway. Mol Med Rep 17:3877-3883 (2018). PubMed: 29359784

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-2 of 2 Abreviews or Q&A

    ELISA abreview for Anti-Galactosidase alpha antibody [EP5828(2)]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    ELISA
    Sample
    Human Recombinant protein (EA.hy926)
    Specification
    EA.hy926
    Blocking step
    BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 21°C
    Type
    Sandwich (Detection)
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Sep 02 2019

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

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK293T)
    Gel Running Conditions
    Reduced Denaturing (gel 10%)
    Loading amount
    10 µg
    Treatment
    transfected
    Specification
    HEK293T
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Mar 07 2018

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