Hi there,

I would prepare enzyme stock in a buffered solution rather than water which H may vary a lot.

The goal is to compare different reactions then the volume and composition of the reaction mixtures have to be identical (apart from the substrate point of view). If DMSO is introduced with your sample the the same amount of DMSO has to be present in the other 2 reaction mixtures.

You intend to perform endpoint kinetics but it is important to know the shape of the kinetics between time 0 and 2h (variation of absorbance in time). For instance in the attached graphics, blue and pink kinetics both reached the same endpoint but obviously pink reaction has been much faster than blue. So endpoint alone is useless. So to evaluate blue versus pink, one relies on the determination of initial velocity which is the slope of the curves at origin.

The other solution is to be sure that after two hours of incubation, all the kinetics will still be in their linear part. So actually the reading will give you the velocity of the reaction as variation of absorbance measured in 2 hours.

 Dear Dominique Liger,

Thank you for your reply. 

I have not initiated yet the assay in my lab. so i have very less knowledge about the kinetics ot of the assay. 

You have suggested to dissolve the enzyme in buffer. What buffer would be suitable for that?

I have plant extracts to check for the lipase inhibitory test. What sort of problems do we face while checking the lipase inhibitory test of plant extracts? 

how is this protocol

I think this found on the net will help:

http://www.agr.hokudai.ac.jp/fbc/lab_manual_files/pancreatic_lipase.pdf

If sample to test is a crude extract then inhibitory effect could be due to indirect effect on lipase activity (for instance substrate binding to molecules in the sample preventing it to be hydrolyzed by the enzyme or presence of lipase natural product which will tend to inhibit the hydrolysis activity).

Dear Dominique Liger,

thank you for the response. i went through your link and read the protocol. It was fine but as our lab is very general lab, we dont have such complex reagents lab. we just want a simple method for lipase inhibition test. would you help to provide some simple and reliable method. if our sample show promising result then we can move to complex method. thank you