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    procathepsin-d-antibody-epr3054-ab134169.pdf

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Neuroscience Cell Adhesion Proteins Membrane Proteins
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-proCathepsin D antibody [EPR3054] (ab134169)

  • Datasheet
  • SDS
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Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Flow Cytometry (Intracellular) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Immunocytochemistry/ Immunofluorescence - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • OI-RD Scanning - Anti-proCathepsin D antibody [EPR3054] (ab134169)
  • Anti-proCathepsin D antibody [EPR3054] (ab134169)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR3054] to proCathepsin D
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-proCathepsin D antibody [EPR3054]
    See all proCathepsin D primary antibodies
  • Description

    Rabbit monoclonal [EPR3054] to proCathepsin D
  • Host species

    Rabbit
  • Tested applications

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

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human proCathepsin D aa 1-100. The exact sequence is proprietary.

  • Positive control

    • Human breast ductal infiltrating carcinoma tissue; A431, MCF7 and SKBR3 cell lysates.
  • 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 -20°C. Stable for 12 months at -20°C.
  • Dissociation constant (KD)

    KD = 1.16 x 10 -10 M
    Learn more about KD
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol (glycerin, glycerine), 0.05% BSA, 59% PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR3054
  • Isotype

    IgG
  • Research areas

    • Neuroscience
    • Cell Adhesion Proteins
    • Membrane Proteins
    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Alzheimer's disease
    • Proteases
    • Signal Transduction
    • Cytoskeleton / ECM
    • Extracellular Matrix
    • ECM Enzymes
    • Other Enzymes
    • Cancer
    • Invasion/microenvironment
    • Angiogenesis
    • ECM enzymes
    • Cathespins
    • Cancer
    • Invasion/microenvironment
    • ECM
    • Extracellular matrix
    • Other
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteolytic enzymes
    • Cysteine protease
    • Cathepsins
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteolytic enzymes
    • Other proteases

Associated products

  • Alternative Versions

    • Anti-proCathepsin D antibody [EPR3054] - BSA and Azide free (ab206472)
  • Assay kits

    • Cathepsin D Inhibitor Assay Kit (ab126779)
    • Cathepsin D Activity Assay Kit (Fluorometric) (ab65302)
  • Isotype control

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

    • Human CTSD knockout A-431 cell line (ab261891)
  • KO cell lysates

    • Human CTSD knockout A-431 cell lysate (ab261700)
  • Positive Controls

    • A-431 whole cell lysate (ab7909)
  • Recombinant Protein

    • Recombinant Mouse Cathepsin D protein (ab191943)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab134169 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)
Use at an assay dependent concentration.
WB
1/1000 - 1/10000. Predicted molecular weight: 44 kDa.
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100.
Notes
Flow Cyt (Intra)
Use at an assay dependent concentration.
WB
1/1000 - 1/10000. Predicted molecular weight: 44 kDa.
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100.

Target

  • Function

    Acid protease active in intracellular protein breakdown. Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease.
  • Involvement in disease

    Defects in CTSD are the cause of neuronal ceroid lipofuscinosis type 10 (CLN10) [MIM:610127]; also known as neuronal ceroid lipofuscinosis due to cathepsin D deficiency. A form of neuronal ceroid lipofuscinosis with onset at birth or early childhood. Neuronal ceroid lipofuscinoses are progressive neurodegenerative, lysosomal storage diseases characterized by intracellular accumulation of autofluorescent liposomal material, and clinically by seizures, dementia, visual loss, and/or cerebral atrophy.
  • Sequence similarities

    Belongs to the peptidase A1 family.
  • Cellular localization

    Lysosome. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Target information above from: UniProt accession P07339 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 1509 Human
    • Omim: 116840 Human
    • SwissProt: P07339 Human
    • Unigene: 654447 Human
    • Alternative names

      • CATD_HUMAN antibody
      • cathepsin D (lysosomal aspartyl protease) antibody
      • Cathepsin D antibody
      • Cathepsin D heavy chain antibody
      • Ceroid-lipofuscinosis, neuronal 10 antibody
      • CLN10 antibody
      • CPSD antibody
      • ctsd antibody
      • EC 3.4.23 antibody
      • EC 3.4.23.5 antibody
      • Lysosomal aspartyl peptidase antibody
      • Lysosomal aspartyl protease antibody
      • MGC2311 antibody
      • OTTHUMP00000042282 antibody
      • OTTHUMP00000196039 antibody
      • OTTHUMP00000198692 antibody
      see all

    Images

    • Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution

      Lane 1 : Wild-type A431 whole cell lysate
      Lane 2 : Cathepsin D knockout A431 whole cell lysate
      Lane 3 : HepG2 whole cell lysate
      Lane 4 : MCF7 whole cell lysate

      Lysates/proteins at 40 µg per lane.

      Predicted band size: 44 kDa
      Observed band size: 46 kDa why is the actual band size different from the predicted?



      Lanes 1 - 4: Merged signal (red and green). Green - ab134169 observed at 46 kDa. Red - loading control, ab7291 (mouse anti-tubulin), observed at 50 kDa.

      ab134169 was shown to recognize in wild-type A431 cells as signal was lost at the expected MW in CTSD knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CTSD knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab134169 and ab7291 (Mouse anti-tubulin loading control) were incubated overnight at 4°C at 1/2000 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-proCathepsin D antibody [EPR3054] (ab134169)
      Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution (purified)

      Lane 1 : MCF-7 cell lysate
      Lane 2 : A431 cell lysate
      Lane 3 : SKBR-3 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 44 kDa
      Observed band size: 44 kDa



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Flow Cytometry (Intracellular) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Flow Cytometry (Intracellular) - Anti-proCathepsin D antibody [EPR3054] (ab134169)

      Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling proCathepsin D with purified ab134169 at 1/100 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

       

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)

      Immunohistochemical staining of paraffin embedded human breast carcinoma with purified ab134169 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    • Immunocytochemistry/ Immunofluorescence - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Immunocytochemistry/ Immunofluorescence - Anti-proCathepsin D antibody [EPR3054] (ab134169)

      Immunofluorescence staining of MCF7 cells with purified ab134169 at a working dilution of 1 in 50, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti rabbit (ab150077), used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab134169 was used at a dilution of 1/200 followed by an Alexa Fluor® 594 goat anti-mouse antibody at a dilution of 1/500.

    • Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Western blot - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      All lanes : Anti-proCathepsin D antibody [EPR3054] (ab134169) at 1/2000 dilution (unpurified)

      Lane 1 : MCF7 cell lysate
      Lane 2 : A431 cell lysate
      Lane 3 : SKBR3 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Standard HRP labelled goat anti-rabbit at 1/2000 dilution

      Developed using the ECL technique.

      Predicted band size: 44 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-proCathepsin D antibody [EPR3054] (ab134169)

      Immunohistochemical analysis of paraffin-embedded Human breast ductal infiltrating carcinoma tissue, staining proCathepsin D using unpurified ab134169 at a 1/250 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • OI-RD Scanning - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      OI-RD Scanning - Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Equilibrium disassociation constant (KD)
      Learn more about KD

      Click here to learn more about KD
    • Anti-proCathepsin D antibody [EPR3054] (ab134169)
      Anti-proCathepsin D antibody [EPR3054] (ab134169)

    Protocols

    • Flow cytometry 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 (1)

    Publishing research using ab134169? Please let us know so that we can cite the reference in this datasheet.

    ab134169 has been referenced in 1 publication.

    • Yang SY  et al. Glucocerebrosidase activity, cathepsin D and monomeric a-synuclein interactions in a stem cell derived neuronal model of a PD associated GBA1 mutation. Neurobiol Dis 134:104620 (2020). PubMed: 31634558

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