Determining absolute protein concentration

[MINA J.G.M.]

Dear All,

 

I am working with a trans membrane protein in the form of microsomal membranes preparation.

I can determine the total protein concentration using Lowry Method.

I also have a specific fluoreometric enzyme assay for my protein.

 

Is there any kinetic/mathematical equation which I can use based on my specific assay to detemine the actual concentration of my active protein in MOLAR CONCENTRATION (c.f titration of the active site)?

 

Thanks in advance,

 

John

[matthijskol]

Dear John,

Do you use overexpression, or endogenous activity of the protein? If
overexpressed, you may be able to compare the activity of your mu
fraction to one where there is no overexpression? If endogenous, can
you perform the assay in a ko/ kd background?

Good luck, Matthijs