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enzyme assay optimization - (Feb/08/2010 )

Hi,

I want to ask opinion from anyone of you who has experience in enzyme assay work. Iíve done optimization for protein concentration and incubation time. The product formation turns to be maximum at 1 mg/ml protein and 30 minutes incubation time. There is small increase in product formation when the protein concentration is more than 1 mg/ml and incubation time is more than 30 minutes. The graph starts to be plateau. My question is, is it correct to pick 1 mg/ml and 30 minutes? One of my friends told me that I should pick any value before it reach max value ie; 0.5 mg/ml and 15 minutes. Which one is correct?

Kentz :D

-kentz19-

what is it that you are trying to determine?

-mdfenko-

mdfenko on Feb 9 2010, 02:57 AM said:

what is it that you are trying to determine?


I'm conducting UGT enzyme assay using p-nitrophenol as a substrate. The substrate is yellow in color and will turn pale yellow when the reaction occur, indicating that the substrate converted to p-NP glucuronide.

Am I right to choose the incubation time and protein conc value?

Thanx

-kentz19-

kentz19 on Feb 9 2010, 11:20 AM said:

mdfenko on Feb 9 2010, 02:57 AM said:

what is it that you are trying to determine?


I'm conducting UGT enzyme assay using p-nitrophenol as a substrate. The substrate is yellow in color and will turn pale yellow when the reaction occur, indicating that the substrate converted to p-NP glucuronide.

Am I right to choose the incubation time and protein conc value?

Thanx



I think that the question should be :

"Are you looking at Agonists or Antagonists of the enzyme". Is it for drug discovery work?

Could we have some more information please!

Kindest regards

Rhombus

-rhombus-

rhombus on Feb 9 2010, 09:23 PM said:

kentz19 on Feb 9 2010, 11:20 AM said:

mdfenko on Feb 9 2010, 02:57 AM said:

what is it that you are trying to determine?


I'm conducting UGT enzyme assay using p-nitrophenol as a substrate. The substrate is yellow in color and will turn pale yellow when the reaction occur, indicating that the substrate converted to p-NP glucuronide.

Am I right to choose the incubation time and protein conc value?

Thanx



I think that the question should be :

"Are you looking at Agonists or Antagonists of the enzyme". Is it for drug discovery work?

Could we have some more information please!

Kindest regards

Rhombus


Im screening medicinal plants ability to inhibit the UGT enzyme...I hope this information will help :)

Thanx

-kentz19-

you can work at just saturating conditions but it may be best to work at half saturating conditions when determining inhibition kinetics.

-mdfenko-

mdfenko on Feb 10 2010, 03:43 AM said:

you can work at just saturating conditions but it may be best to work at half saturating conditions when determining inhibition kinetics.


Dear mdfenko,

May I know why?

kentz19

-kentz19-

so that if the inhibitor is competitive then the conditions won't overwhelm the inhibition.

-mdfenko-