| Assay Method Information | |
| | HtrA1 Enzyme Inhibition Assay |
| Description: | Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore, whose emission is quenched in the intact peptide.Assay buffer: 500 mM Tris pH 8.0, 200 mM NaCl, 0.025% CHAPS, 0.005% BSGEnzyme: human HtrA1 Cat-PDZ, final concentration 1 nMSubstrate: Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys (SEQ ID NO: 1), final concentration 500 nM (from Innovagen Cat: SP-5076-1, Lot: 89584.02)Mca=(7-Methoxycoumarin-4-yl)acetylDnp=2,4-DinitrophenylFinal volume: 51 μlExcitation 320 nm, emission 390 nmAfter a pre-incubation of the HtrA1 protease for 30 min with compounds, substrate is added to the wells and initial RFU is measured. Upon incubation for 2 hours at RT, the enzymatic activity cleaved the substrate releasing fluorescent Mca-peptide conjugate and the final RFU value is measured. The presence of inhibitors leads to a decreased final RFU.For the analysis ΔRFU is calculated as RFUend−RFUstat and then percent inhibition is calculated with the following formula:PCT_Inhibition=100-100*(ΔRFU compound −ΔRFU blank)/(ΔRFU negctrl −ΔRFU blank)whereneg.ctrl is protease with substrate and DMSOblank is as neg. ctrl without proteasecompound is as neg. ctrl with test compounds at desired concentrationThe IC50 is determined using a 4-point Hill-fit equation wherex=concentration of test compoundA=extrapolated value of the curve at effector concentration equals 0B=extrapolated value of the curve at effector concentration equals infiniteC=concentration at the inflection point of the sigmoidal curve (IC50)D=Hill coefficient of slope at the inflection point of the fitted curveY ( x ) = A + B - A D 1 + ( C x )As a counter screen the compounds are added to the protease-substrate reaction mix only after 2 h incubation, when all the substrate is turned over, to identify auto-fluorescent or absorbing compounds giving false positive hits. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |