565785 β-Secretase Activity Assay Kit, Fluorogenic

565785
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorogenic

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      565785-1KIT
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          Description
          OverviewA sensitive fluorogenic assay kit for the determination of β-secretase (BACE) activity (Excitation max: 335-355 nm; Emission max: 495-501 nm). β-Secretase is a transmembrane aspartyl protease that cleaves membrane-bound amyloid precursor protein.
          Catalogue Number565785
          Brand Family Calbiochem®
          SynonymsBACE Activity Assay Kit, Fluorogenic
          Application Data
          The activity of recombinant was measured using the β-Secretase Substrate, &alpha-secretase substrate (not included), and γ-secretase substrate (not included) as outlined in the Detailed Protocol above.
          References
          Product Information
          Detection methodFluorogenic
          Form100 Tests
          FormatCuvette or 96-well plate
          Kit containsExtraction Buffer, Reaction Buffer, β-Secretase Substrate, β-Secretase Protein (Positive Control), β-Secretase Inhibitor, and a user protocol.
          Applications
          Biological Information
          Assay time1.5 h
          Sample TypeCell lysates, tissue extracts, purified BACE
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Intended useThe Calbiochem® β-Secretase Activity Assay Kit, Fluorogenic, is intended for measuring the β-secretase (BACE) activity in cell lysates, tissue extracts, or purified enzyme preparations.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
          Storage -20°C
          Storage ConditionsUpon arrival store the entire contents of the kit at -20°C. Following reconstitution of the Active β-Secretase, aliquot and freeze (-70°C) to avoid loss of activity. Following initial thaw of the kit contents, store the Extraction Buffer and 2X Reaction Buffer at 4°C.
          Protect from Light Protect from light
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsExtraction Buffer, Reaction Buffer, β-Secretase Substrate, β-Secretase Protein (Positive Control), β-Secretase Inhibitor, and a user protocol.
          Specifications

          Documentation

          β-Secretase Activity Assay Kit, Fluorogenic SDS

          Title

          Safety Data Sheet (SDS) 

          β-Secretase Activity Assay Kit, Fluorogenic Certificates of Analysis

          TitleLot Number
          565785
          User Protocol

          Revision22-May-2017 JSW
          SynonymsBACE Activity Assay Kit, Fluorogenic
          Form100 Tests
          FormatCuvette or 96-well plate
          Detection methodFluorogenic
          StorageUpon arrival store the entire contents of the kit at -20°C. Following reconstitution of the Active β-Secretase, aliquot and freeze (-70°C) to avoid loss of activity. Following initial thaw of the kit contents, store the Extraction Buffer and 2X Reaction Buffer at 4°C.
          Intended useThe Calbiochem® β-Secretase Activity Assay Kit, Fluorogenic, is intended for measuring the β-secretase (BACE) activity in cell lysates, tissue extracts, or purified enzyme preparations.
          Backgroundβ-Secretase has been identified as an excellent target for anti-amyloid therapy in the treatment of Alzheimer's disease.
          Principles of the assayThe Calbiochem® β-Secretase Activity Assay Kit, Fluorogenic, provides a convenient fluorescence method for detecting β-secretase activity in cell lysates and purified samples. The assay utilizes a secretase-specific peptide conjugated to EDANS and DABCYL. When the substrate is intact (i.e., uncleaved) the fluorescence from EDANS is effectively quenched by the DABCYL moiety. Cleavage of the peptide by β-secretase physically separates EDANS and DABCYL, allowing the emission of the fluorescence signal. The increase in fluorescence is measured at an excitation wavelength of 335-355 nm and an emission wavelength of 495-510 nm.
          Materials provided• Extraction Buffer (Kit Component No. KP31430): 1 bottle, 25 ml
          • 2X Reaction Buffer (Kit Component No. KP31431): 1 vial, 10 ml
          • β-Secretase Substrate (Kit Component No. KP31432): 1 vial, 200 µl, supplied in DMSO
          • Active β-Secretase (Kit Component No. KP31433): 1 vial, liquid
          • β-Secretase Inhibitor (Kit Component No. KP31434): 1 vial, 10 µl, supplied in DMSO
          Reagent preparation• Active β-Secretase: Reconstitute the lyophilized Active β-Secretase with 10 µl ddH2O. Following reconstitution, aliquot and freeze (-70°C) to avoid loss of activity.
          Detailed protocol1. Treat cells as needed prior to preparation of cell lysates. A parallel sample without any treatment should be prepared to assess the background level of activity.
          2. Collect cells (allow ~2-5 x 106 cells or 25-200 µg total protein per assay) by centrifugation for 5 min at 700 x g.
          3. Resuspend the cells in 100 µl ice-cold Extraction Buffer. Incubate on ice for 10-20 min. For tissue samples, add 2-3X the volume of ice-cold Extraction Buffer and homogenize at 4°C.
          4. Centrifuge for 1 min in a microcentrifuge (10,000 x g), transfer the supernatant to a fresh tube, and place on ice.
          5. Add up to 50 µl cell lysate or tissue extract to designated wells in a 96-well plate that is suitable for measurement in a fluorescence plate reader. If the sample volume is less than 50 µl, adjust the final volume to 50 µl with Extraction Buffer. For a positive control, mix 2 µl Active β-Secretase with 48 µl Extraction Buffer in designated control wells. For a background control, mix 49 µl Extraction Buffer with 49 µl 2X Reaction Buffer and 2 µl β-Secretase Substrate. Untreated cell lysate can be used to assess basal level of cellular enzymatic activity. β-Secretase Inhibitor (2 µl) can be added to the purified enzyme or treated cell lysate to assess specificity.
          6. Add 48 µl 2X Reaction Buffer and 2 µl β-Secretase Substrate to each well. Tap gently to mix. Alternatively, the reaction can be carried out in a microfuge tube if a fluorescence plate reader is not available.
          7. Incubate at 37°C for 1-2 h in the dark.
          8. Read the fluorescence of the samples in a fluorescence plate reader using an excitation wavelength of 335-355 nm and an emission wavelength 495-510 nm. Alternatively, the samples can be transferred to a quartz microcuvette and read in a fluorimeter if a fluorescence plate reader is not available.
          9. Background reading from substrate and buffers (without secretase activity) must be subtracted from all samples prior to data analysis. Due to the nature of the fluorescence quenching substrate, the background reading is significant. The changes in β-secretase activity can be determined by comparing readings from treated and untreated samples and the result can be presented as fold increase. Alternatively, the activity can be expressed as Relative Fluorescent Unit (RFU) per sample, cell numbers, or amount of protein.
          Example data

          Figure 1: Activity of Recombinant β-Secretase

          The activity of recombinant was measured using the β-Secretase Substrate, &alpha-secretase substrate (not included), and γ-secretase substrate (not included) as outlined in the Detailed Protocol above.

          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals Inc.