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Description
Overview
A 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 Number
565785
Brand Family
Calbiochem®
Synonyms
BACE 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 method
Fluorogenic
Form
100 Tests
Format
Cuvette or 96-well plate
Kit contains
Extraction Buffer, Reaction Buffer, β-Secretase Substrate, β-Secretase Protein (Positive Control), β-Secretase Inhibitor, and a user protocol.
The 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 Conditions
Upon 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 contains
Extraction Buffer, Reaction Buffer, β-Secretase Substrate, β-Secretase Protein (Positive Control), β-Secretase Inhibitor, and a user protocol.
β-Secretase Activity Assay Kit, Fluorogenic Certificates of Analysis
Title
Lot Number
565785
User Protocol
Revision
22-May-2017 JSW
Synonyms
BACE Activity Assay Kit, Fluorogenic
Form
100 Tests
Format
Cuvette or 96-well plate
Detection method
Fluorogenic
Storage
Upon 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 use
The 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.
Background
β-Secretase has been identified as an excellent target for anti-amyloid therapy in the treatment of Alzheimer's disease.
Principles of the assay
The 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.
• 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 protocol
1. 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 Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals Inc.
