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Beta-Secretase Activity Fluorometric Assay Kit Kit

AcA Reactivity: Mammalian Fluorometric Cell Lysate, Plasma, Serum, Tissue Samples
Pubmed (2)
Catalog No. ABIN411640
$525.00
Plus shipping costs $45.00
100 tests
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Delivery in 2 to 3 Business Days
  • Target
    beta Secretase
    Reactivity
    Mammalian
    Detection Method
    Fluorometric
    Application
    Activity Assay (AcA)
    Sample Type
    Cell Lysate, Plasma, Serum, Tissue Samples
    Specificity
    The beta -Secretase activity Assay Kit provides a convenient fluorescence method for detecting beta -secretase activity in biological and purified samples. The assay utilizes a secretase-specific peptide conjugated to two reporter molecules EDANS and DABCYL. In the uncleaved form, the fluorescent emissions from EDANS are quenched by the physical proximity of the DABCYL moiety. Cleavage of the peptide by secretase physically separates EDANS and DABCYL allowing for the release of a fluorescent signal. The level of secretase enzymatic activity in samples is proportional to the level of fluorescence intensity.
    Characteristics
    β-Secretase Activity Assay Kit: convenient Fluorometric Assay for Detecting β-secretase Activity in Biological and Purified samples.
    Components
    β-Secretase Extraction Buffer
    β-Secretase Reaction Buffer (2X)
    β-Secretase Substrate (in DMSO)
    Active β-Secretase (Lyophilized)
    β-Secretase Inhibitor (in DMSO)
  • Application Notes
    Convenient fluorescence method for detecting β-secretase activity in biological and purified samples.
    Comment

    Further details regarding sample type: Cell and tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids

    Protocol
    A. General Consideration & Reagent Preparation: Reconstitute the lyophilized Active beta -Secretase to 10 µL ddH2O. The enzyme should be refreezed immediately at -70 °C after each use to avoid loss of activity. The enzyme is sufficient for 5 positive control assays (2 µL/assay). Assay can be performed directly in a 96-well plate. Nunc F16 Black MaxiSorp TM polystyrene microplate is recommended. B.
    1. Collect cells (5 x 10^6 cells/assay) by centrifugation for 5 min at 700x g. Add 0.1 mL of ice-cold Extraction Buffer. For tissue sample, add 2-3X volume of ice-cold Extraction Buffer to tissue sample and homogenize it on ice.
    2. Incubate cell lysate on ice for 10 minutes and centrifuge at 10,000x g for 5 minutes. Transfer the supernatant to a new tube and keep on ice. This should yield a lysate with a protein concentration of approx. 2-4 mg/mL.
    3. Add 50 µL cell lysate (approx. 2 - 5 x 10^6 cells or 25 - 200 µg of total protein) to each well in a 96-well plate. For positive control assay, add 2 µL of reconstituted Active beta -secretase to 50 µL of Extraction Buffer. For negative control assay, add 2 µL of the beta -Secretase Inhibitor to 50 µL of Extraction Buffer.
    4. Add 50 µL of 2X Reaction Buffer.
    5. Add 2 µL of beta -Secretase substrate.
    6. Cover the plate, tap gently to mix, and incubate in the dark at 37 °C for 1-2 hours.
    7. Read sample in a fluorescence plate reader with Ex. = 335-355 nm and Em. = 495-510 nm. Background reading from substrate (without secretase) must be subtracted from all treated and untreated samples before calculating the fold increase in secretase activity (Note: Background reading from substrate can be quite high, due to the nature of such fluorescence quenching assay.) beta -Secretase activity can be expressed as the Relative Fluorescence Units per µg of protein sample.
    Restrictions
    For Research Use only
  • Storage
    -20 °C
    Expiry Date
    12 months
  • Gamerdinger, Clement, Behl: "Cholesterol-like effects of selective cyclooxygenase inhibitors and fibrates on cellular membranes and amyloid-beta production." in: Molecular pharmacology, Vol. 72, Issue 1, pp. 141-51, 2007 (PubMed).

    Kern, Roempp, Prager, Walter, Behl: "Down-regulation of endogenous amyloid precursor protein processing due to cellular aging." in: The Journal of biological chemistry, Vol. 281, Issue 5, pp. 2405-13, 2006 (PubMed).

  • Target
    beta Secretase
    Alternative Name
    beta-Secretase
    Background
    β-Secretase has been implicated to be an excellent target for anti-amyloid therapy for the treatment of Alzheimer's disease. The β-Secretase activity Assay Kit provides a convenient fluorescence method for detecting β-secretase activity in biological and purified samples. The assay utilizes a secretase-specific peptide conjugated to two reporter molecules EDANS and DABCYL. In the uncleaved form, the fluorescent emissions from EDANS are quenched by the physical proximity of the DABCYL moiety. Cleavage of the peptide by secretase physically separates EDANS and DABCYL allowing for the release of a fluorescent signal. The level of secretase enzymatic activity in samples is proportional to the level of fluorescence intensity.
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