I want to ask for the activity measurement method of BACE1 Protein, Human (HY-P72840).
I bought the recombinant protein of MCE. But when I made it myself, the fluorescence signal was very weak. It was the same when I changed the substrate Mca-YEVD-AMC. The background was so high that I could hardly see the difference. I suspected that I did it wrong.
Points I want to ask:
- Optimal reaction system: Is there a recommended buffer formula (such as containing 0.1% CHAPS to help solubilize)? What protein concentration is generally used?
- Substrate details: Concentration range of fluorescent substrate (50 μM given in the manual, is it too low?), do I need to pre-incubate the substrate and protein?
- Detection skills: When reading with a fluorescent microplate reader, does the plate need to be protected from light? How to solve the high background (I tried blank without adding protein, the signal is still high, is it the substrate itself that decomposes?)
- Pitfall avoidance experience: Have you encountered the problem of poor protein solubility? Do I need to use 0.01% Tween-20 to prevent adsorption? How long can the activated protein be stored at 4°C?
Measured by its ability to cleave a fluorogenic peptide substrate, Mca-SEVNLDAEFRK(Dpn)RR-NH2 (Cat.NO.: HY-P1859).
Read at excitation and emission wavelengths of 320 nm and 405 nm.
The method we tested, specific activity is 1.79 pmol/min/µg, as measured under the described conditions.
Materials in details：
Assay Buffer: 0.1 M Sodium Acetate, pH 4.0
Recombinant Human BACE 1 (Catalog HY-P72840)
Fluorogenic Peptide Substrate : MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DPN)-Arg-Arg-NH2 (Catalog HY-P2714)
F16 Black Maxisorp Plate
Fluorescent Plate Reader or equivalent
Assay Protocol：
Calculate specific activity:
Specific Activity (pmol/min/µg) = Adjusted Vmax* (RFU/min) x Conversion Factor* (pmol/RFU) x amount of enzyme (µg)
Note:
The answer to this question comes from MCE Technical Support. How to contact us?
- Tel:609-228-6898
- Fax:609-228-5909
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- Address:1 Deer Park Dr, Suite F, Monmouth Junction, NJ 08852, USA
NH2-RE(EDANS)EVNLDAEFK(Dabcyl)R-COOH is the substrate we use for B-secretase activity determination. Using 2 ul of a 500 uM stock per well. I have attached a typical result showing background , enzyme only and enzyme + inhibitor
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