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RE: What is an easy way to determine the active fraction of a recombinantly expressed enyzme prep?

in #stemq5 years ago (edited)

This just answers a question of how I might quantitate product. However that is not the question I am asking!

The question I am asking is, what sort of reaction would I need to set up to determine the active fraction of the enzyme. A serial dilution of the enzyme can tell me whether or not my enzyme behaves normally (IE we should see a linear relationship between enzyme concentration and product formation rate). It also potentially allows me to calculate specific activity (nmol product/min/mg protein) and that should be consistent no matter how much enzyme I include. However, none of that tells me how much of that enzyme is actually turning over, it just tells me whether the same amount relative to how much there is total, is turning over, and there are no concentration based effects (oligomerization of enzyme changing activity etc..).

You're right a good Enzyme Kinetics book could help with that part, a particular type of kinetic experiment could answer this question well (hint, look at the plot).

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Ah...this is some kind of quiz. I thought you needed help. I am not so interested now, hahahaha. However, the plot appears to fit Michaelis Menten kinetics.

I don't need help with enzyme kinetics ;)

This is a StemQ question, I am hoping someone will answer it. It's not a quiz. I know the answer, but I'm not making a post to provide it.

The plot is not of the appropriate relationship for MM kinetics. :)

It's not steady state (at least not exclusively.)

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