Store Kit at 4-10°C
***DO NOT FREEZE***
*******************
™
EnSens® MMP-14 Activity Detection Kit
(Cat. No. 032014_03_25, 032014_03_96, or 032014_03_384)
Fluorescence Based Activity Assay for Microplates
Note: Your kit is shipped with an ice
pack; if your kit arrives at room
temperature, no worries. Kit
components are stable at room
temperature for several days.
Kit Components:
 EnSens® MMP-14 Activity Detection Substrate (stored in 50mM Tris-Cl pH 7.5,
750mM NaCl, 50mM imidazole, 0.1% TritonTM X-100, 0.02% TweenTM 20), 2µM
 Far Red Fluorogen (FRF dye), 2µM
 Pre-activated substrate (EnSens®-PA, substrate positive control), 2µM*
 2X Assay Buffer
 Molecular biology grade water
Other Required Components:
 Active protease** (for quantitative standard curve)
 Microplate (black plate with round- or flat-bottom wells is appropriate)
 Plate reader with fluorescence detection capabilities
Fluorescence Detection:
Excitation/Emission: 635 nm (625-635 nm) / 656 nm (655-665 nm)
Depending on your fluorimeter, the gain/sensitivity setting may need to be increased
manually, and the bandwidth decreased to 5-10nm. Contact us at [email protected]
with questions.
Suggested Assay Conditions: 50 uL final volume is recommended for 96-well plates
and 20 uL volume for 384-well plates. Volume can be scaled from 10 - 500μL;
temperature between 4°C and 37°C; assay time can range from 10 minutes to several
hours, depending on concentration and activity of protease in your sample.
Example Protocol for 5 μL Sample in 50 μL total volume (25-well and 96-well kits):
In microplate well or microfuge tube:
Mix 25 μL 2X Assay Buffer
5 μL EnSens® Substrate or EnSens®-PA substrate positive control
5 μL Far Red Fluorogen
10 μL Water
Mix thoroughly
Incubate at RT or 4°C for 15-60 minutes protected from light
Add 5μL of your sample containing protease activity***
Mix thoroughly. Read fluorescence at Ex/Em 625-635 nm/655-665 nm
*To ensure accurate reads, always set your fluorimeter gain using the EnSens ®-PA, substrate positive control, while
keeping in mind that the EnSens®-PA signal will be very strong relative to the signal in your sample wells.
**A standard curve of active protease must be run each time a plate is read in order for the assay to yield quantitative
results.
***Always add your sample last, after the 15 minute incubation of Buffer:EnSens:Far Red Fluorogen mix. Adjust
sample and water volume as needed. For best results, use a range of sample dilutions and perform all assays in
duplicate or triplicate. Please see Fluorimetry Assay Optimization Suggestions included in this insert.
Enzium, Inc.
www.Enziumlabs.com
Technical Support: [email protected]
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Kit Components Exclusively Licensed from Carnegie Mellon University and Sharp Edge Labs
Rev.12May2015