how to convert RFU/sec of my Vmax in mol/min/mg of protein??
Posted 15 February 2013 - 05:50 AM
I'm doing some enzymatic kinetic studies and I need your help!!I'm using a FRET based assay to evaluate the strenght of some potential inhibitors against HIV-1 protease.
I wanted to measure the Km and the Vmax of HIV-1 protease for my peptidic substrate http://www.sigmaaldr...atalog/product/sigma/h0790?lang=it®ion=IT and I performed different enzymatic reaction at 8 substrate concentrations. I used graphpad to obtain Km and Vmax, but the Vmax is expressed as RFU/sec and I know I should convert it as mol/min/mg of protein. How can I do this?
I think I should have a calibration curve of my product but I'm not sure...
I tried to perform a calibration curve using the anthranilic acid, but at the end the concentration I obtained was negative....Where is the mistake????
thanks a lot
Posted 18 February 2013 - 03:18 AM
Edited by neuron, 18 February 2013 - 03:20 AM.
Posted 19 February 2013 - 01:02 AM
Posted 19 February 2013 - 03:04 AM
Posted 19 February 2013 - 03:21 PM
Don't too concerned with the absolute values of the standard curve, rather just focus on the rate of the change. An increase in x uM product causes an increase in fluorescent signal of y; therefore when the enzyme causes a change in fluorescence of y per min the rate will be x uM/min.
Fluorescent readings can be susceptible to their microenvironment, which is the basis of FRET assays, so ideally you would use the peptide fragment to generate the standard curve but I suspect that it may not be readily available so your approach will be acceptable. It might be possible to check by letting the reaction proceed to completion and hence checking if the change in fluorescence is about the same as you would calculate from the standard curve for a given amount of substrate.