wise to do an enzyme kinetic assay with unpurified enzyme? - (Jun/28/2013 )

HOYAJM on Mon Aug 19 18:15:49 2013 said:

You can not use unpurified enzymes for kinetics. One of the most crucial aspects of an kinetic assay for an enzyme is , or enzyme concentration. You will be only guessing what the concentration of your unpurified enzyme is. Thus, your kcat value would be way off and not reliable.

as i pointed out in an earlier post, you can do preliminary work on impure enzyme. kcat is not the only kinetic parameter that may be determined (substrate saturation, inhibitor kinetics, etc). also, you may be able to determine percent of total for your protein of interest at any stage of purification so you can make a better guess of the enzyme concentration.

you would, of course, want to perform kinetics with purified enzyme after.

mdfenko on Tue Aug 20 04:32:49 2013 said:

 

HOYAJM on Mon Aug 19 18:15:49 2013 said:

You can not use unpurified enzymes for kinetics. One of the most crucial aspects of an kinetic assay for an enzyme is , or enzyme concentration. You will be only guessing what the concentration of your unpurified enzyme is. Thus, your kcat value would be way off and not reliable.

as i pointed out in an earlier post, you can do preliminary work on impure enzyme. kcat is not the only kinetic parameter that may be determined (substrate saturation, inhibitor kinetics, etc). also, you may be able to determine percent of total for your protein of interest at any stage of purification so you can make a better guess of the enzyme concentration.

 

you would, of course, want to perform kinetics with purified enzyme after.

 

Yes you did. I missed that. All of my enzyme kinetics have been with FRET peptides ($750 for a few mg's) and at that price, I just never bothered until I had pure enzyme. But, if your assays are cheap, then I guess it doesnt hurt to use a crude prep for a trial.