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Cathepsin D Activity Fluorometric Assay Kit Kit

AcA Reactivity: Mammalian Fluorometric Cell Lysate, Tissue Samples
Pubmed (2)
Catalog No. ABIN411881
$425.00
Plus shipping costs $45.00
100 tests
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Delivery in 2 to 3 Business Days
  • Target
    Cathepsin D (CTSD)
    Reactivity
    • 7
    • 6
    • 3
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Mammalian
    Detection Method
    Fluorometric
    Application
    Activity Assay (AcA)
    Purpose
    The Cathepsin-D Activity Assay kit is a fluorescence-based assay that utilizes the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA. Cell lysates or other samples that contain cathepsin-D will cleave the synthetic substrate to release fluorescence, which can then easily be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm.
    Sample Type
    Cell Lysate, Tissue Samples
    Specificity
    The Cathepsin-D Activity Assay kit is a fluorescence-based assay that utilizes the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA. Cell lysates or other samples that contain cathepsin-D will cleave the synthetic substrate to release fluorescence, which can then easily be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm. The cathepsin-D assay is simple, straightforward, and can be adapted to 96-well plate assays. Assay conditions have been optimized to obtain the maximal activity.
    Characteristics
    Cathepsin D Activity Assay Kit: Rapid & Convenient Fluorometric Assay to Measure Cathepsin D Activity within 1-2 hrs.
    Components
    CD Cell Lysis Buffer
    CD Reaction Buffer
    CD Substrate (1mM)
  • Application Notes
    Detects alternative proteolytic enzymes such as the lysosomal cathepsin proteases that can initiate or propagate proapoptotic signals.
    Comment

    Further details regarding sample type: Cell and tissue lysates

    Assay Time
    1 - 2 h
    Protocol
    1. Collect cells (1 x 10^6 ) by centrifugation.
    2. Lyse cells in 200 µL of chilled CD Cell Lysis Buffer. Incubate cells on ice for 10 min.
    3. Centrifuge for 5 min at top speed. Transfer the clear cell lysate into a labeled new tube.
    4. Add 5-50 µL of the cell lysate (or approx. 1-10 ng of purified Cathepsin D protein samples) into each well in a 96-well plate. Bring the total volume to 50 µL with CD Cell Lysis Buffer.
    5. Prepare a master assay mix, for each assay: 50 µL of Reaction Buffer 2 µL of Substrate
    6. Mix the master assay mix. Add 52 µL of the master assay mix into each assay wells. Mix well. Incubate at 37 °C for 1-2 hour.
    7. Read samples in a fluorometer equipped with a 328-nm excitation filter and 460-nm emission filter. Cathepsin D activity can be expressed by the relative fluorescence units (RFU) per million cells, or RFU per microgram protein of your sample, or RFU fold increase of treated samples vs the untreated control or the negative control sample.
    Restrictions
    For Research Use only
  • Storage
    -20 °C
    Expiry Date
    6-12 months
  • Liu, Xu, Ye, Liang, Wu, Zheng, Deng, Li, Liu: "Urinary proteins induce lysosomal membrane permeabilization and lysosomal dysfunction in renal tubular epithelial cells." in: American journal of physiology. Renal physiology, Vol. 308, Issue 6, pp. F639-49, 2015 (PubMed).

    Vitner, Dekel, Zigdon, Shachar, Farfel-Becker, Eilam, Karlsson, Futerman: "Altered expression and distribution of cathepsins in neuronopathic forms of Gaucher disease and in other sphingolipidoses." in: Human molecular genetics, Vol. 19, Issue 18, pp. 3583-90, 2010 (PubMed).

  • Target
    Cathepsin D (CTSD)
    Alternative Name
    Cathepsin D (CTSD ELISA Kit Abstract)
    Synonyms
    catD, fb93e11, fj17b09, wu:fb93e11, wu:fj17b09, LOC398557, Cathd, CatD, LOC100136761, CTSD, DKFZp469J0315, DDBDRAFT_0205785, DDBDRAFT_0215012, DDB_0205785, DDB_0215012, CLN10, CPSD, CD, cathepsin D, cathepsin D S homeolog, lysosomal aspartic protease, cathepsin D-like, ctsd, LOC398557, ctsd.S, CatD, LOC5567565, LOC100136761, CTSD, Cathd, ctsD, catd, Ctsd, LOC443060
    Background
    Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases may initiate or propagate proapoptotic signals. Cathepsins are lysosomal enzymes that are also used as sensitive markers in various toxicological investigations. The Cathepsin-D Activity Assay kit is a fluorescence-based assay that utilizes the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA. Cell lysates or other samples that contain cathepsin-D will cleave the synthetic substrate to release fluorescence, which can then easily be quantified using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm.
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