Beta-gal reporter assay = transcription? - How does this work? (May/07/2008 )
I have a quesiton that hopefully people will be able to answer.
I know people use the beta-gal assay as a reporter for transcription when looking at a promoter. But the beta-gal assay detects the presence of a protein. How does the presence of this protein equate to the rate of transcription for the gene in a quantitative way? Can it? Clearly there has to be a transcript before there is a protein, but the presence of a protein reflects the amount of other proteins that might be in the system, discounting degredation, no? How does it reflect transcription except indirectly? Ackkk! Thanks for the help here. I appreciate it.
As you say, it does not, except indirectly. But the rate of degradation is proportional to the amount of protein, while the rate of production is independent of the amount, so the equilibrium value reflects a measure of the rate of production.
d [protein]/dt = K(transcription) - D[protein] = 0 (in equilibrium)
[protein] = K/D
So, you get a relative measure of K in terms of the degradation coefficient. You can also see the limits -- it depends on the linearity of the degradation process (not overloaded) and on the linearity of the transcription/translation machinery (not overloaded) and the consistency of these processes. That's a lot of "depends on." Also, you have to think about cell growth, since these numbers assume a constant volume. You can think of cell growth as a kind of dilution, increasing D. So, growth rate must be constant (no stationary phase, for this and many other reasons). OD < 0.1 would be good.
And if you are to use such method, you can always measure the transcription by qPCR, witch will give you the exact number of copy or the folds of induction.
Great answer Phage, by the way