Studying genes at the Trancription& Translation level

[Sr_08]

Hi,

I working on a strain that doesn't require a substrate to express the enzyme whereas other strian among the smae species require to. So, I am lil bit confused on how do I go about the whole thing?
Do I have to get the total RNA, do a real time PCR etc;??

[danfive]

Originally Posted by Sr_08

Hi,

I working on a strain that doesn't require a substrate to express the enzyme whereas other strian among the smae species require to. So, I am lil bit confused on how do I go about the whole thing?
Do I have to get the total RNA, do a real time PCR etc;??

If you want to study trancripition and translation levels, that means you want the mRNA and protein levels.

You likely want a quantitative Real-time-RT-PCR (one-step rev-trancription-PCR is pretty nice) and maybe an ELISA or quantitative WB.

Another option for protein-of-interest quantitation, is either immuno-affinity or tag-specific purification, then a simple BCA protein assay of purified protein (with a SDS-gel for purity confirmation, gel quantitation w/ a standard and appropriate software is also possible).

Or if the enzyme activity can be measured, you can measure enzyme activity relative to amount of mRNA per ug sample/#cells (or whatever quantity).