Ratio of labelled vs. unlabelled species in "uptake assay" - What is proper: Using a fixed amount or a fixed concentration of label (Dec/18/2007 )
This might be straightforward, but please enlighten me:
I am doing an assay, where I want to see how (moles) much of a molecular species is taken up by my model organism (C. elegans) at various concentrations of that molecule. I want to use a radiolabelled species, but it is unfeasible to use radiolabelled species only. But which is the proper thing to do:
a: Use the same amount of radiolabelled species in all samples/at all concentrations - and add unlabelled species to the desired final concentration,
b: Prepare a mastermix of labelled and unlabelled species and use that for preparing all samples - meaning that the ratio labelled:unlabelled remains constant in all samples,
c: It doesn't really matter what I do - both approaches are valid.
So, which is it? What would you do?
the answer is c.
i've done experiments both ways (not with c. elegans) and they are both valid. just make sure you calculate correctly.
i, however, prefer b (easier calculation).