Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

how to convert RFU/sec of my Vmax in mol/min/mg of protein??


  • Please log in to reply
4 replies to this topic

#1 claire84

claire84

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 33 posts
2
Neutral

Posted 15 February 2013 - 05:50 AM

Hi everybody!!
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&region=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

Claire

#2 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 179 posts
5
Neutral

Posted 18 February 2013 - 03:18 AM

Yes you need to have a calibration curve that would tell you 1 RFU means how many moles? Let say you got X rfu for Y moles then these y moles will be divided by the time to get you the vmax at 2Km concentration. And calibration curve can be generated with any compound that gives RFU. If you are geting negative readings for some values, may be you are doing something wrong. Or you can omit those values next time when you do it again to generate the calibration curve.

Edited by neuron, 18 February 2013 - 03:20 AM.


#3 claire84

claire84

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 33 posts
2
Neutral

Posted 19 February 2013 - 01:02 AM

But I should use the peptidic fragment the enzyme generate to make the calibration curve, or can I use only the fluorophore (in this case the anthranilic acid)?

#4 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 179 posts
5
Neutral

Posted 19 February 2013 - 03:04 AM

In my view you can use any fluorophore because its relative fluorescence unit. Like for radioactivity assays (for instance c14) we make a calibration curve with the counts generated by any radioactive compound having c14. So basically you need to know How much moles are there in particular RFU to calculate vmax and km.

#5 DRT

DRT

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
7
Neutral

Posted 19 February 2013 - 03:21 PM

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????


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.

But I should use the peptidic fragment the enzyme generate to make the calibration curve, or can I use only the fluorophore (in this case the anthranilic acid)?



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.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.