determining efficiency of RNase treatment - (Nov/21/2006 )
I am using a TNT coupled reticulocyte lysate system to make my favorite protein in vitro. I will be using my favorite protein in a GST pull down assay. We believe my favorite protein interacts with protein X, but its interaction is dependent on the presecne of RNA. The only RNA present in my lysate is that of protein X, in the form of tRNA and mRNA. I want to degrade this RNA by treating with RNase A and see if my favorite protein still interacts with protein X. As a control, I need to show that RNase treatment degraded most if not all of the RNA in my lysate. Does anyone know how I can do this?
not sure if this will help, but perhaps you could do a PCR that would only work on mRNA and not DNA. If you had amplification in the wells treated with RNase A, you have mRNA contamination.