Recombinant 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
Related conjugates and formulations
Overview
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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.
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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
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
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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...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP5828(2) -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
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 |
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Flow Cyt (Intra) |
1/90.
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IP |
1/10 - 1/100.
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WB | (1) |
1/1000 - 1/10000. Predicted molecular weight: 49 kDa.
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IHC-P |
1/50 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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ICC/IF |
1/50 - 1/500.
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Notes |
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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. |
ICC/IF
1/50 - 1/500. |
Target
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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. - Information by UniProt
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Database links
- Entrez Gene: 2717 Human
- Omim: 300644 Human
- SwissProt: P06280 Human
- Unigene: 69089 Human
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Alternative names
- AGAL_HUMAN antibody
- Agalsidase alfa antibody
- Alpha D galactosidase A antibody
see all
Images
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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 kDaLanes 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.
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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).
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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 kDaLanes 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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (8)
ab168341 has been referenced in 8 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
- Guo H et al. Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy. Cell Death Differ 28:320-336 (2021). PubMed: 32764647
- Jehn U et al. α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes. Int J Mol Sci 22:N/A (2021). PubMed: 34768768
- 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