BindingDB logo
myBDB logout

Assay Method Information

Assay Name:  enzyme-based fluorimetric assay
Description:  For this assay, the reagents used as follows. The substrate corresponds to Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg 25 mm in assay buffer.  This low-fluorescent peptide is cleaved by MMPs to provide Mca-Pro-Leu-Gly, which is intensely fluorescent at an excitation at 340 nm and emission at 405 nm. While the enzymes pro-MMP-2 250 pm and MMP-9 (recombinant catalytic sequence) 500 pm in assay buffer. The assay buffer consists of Tris⿿HCl 0.1 m, pH 7.5, NaCl 0.1 m, CaCl2 10 mm, 0.05% v/v BRIJ35, APMA (p-aminophenylmercuric acetate) 1 mm.  This was carried out in 96-well plate, a solution of inhibitor (50 mL) at the appropriate concentration and enzyme (50 mL) was incubated together at 25 °C for 30 min, and then 10 mL of substrate solution was added and the plate was further incubated 3 h at 37 °C. The incubation solutions were quenched with acetic acid (100 mL of 3% soln.) and their fluorescence was recorded with the help of a plate reader spectrometer.  The inhibitory activities were calculated against a control and IC50 curves, derived from 6 different inhibitor concentrations, extrapolated by means of statistical analysis program graphpad prism (v. 4.0; San Diego, CA, USA). Each inhibitor concentration was used in triplicate and the experiments run twice.
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail
   
    

Home

|

Search

|

Deposit

|

SiteMap

|

About us

|

Email us

|

Info

 
Last update November 1, 2007
©2000 BindingDB. All rights reserved.