in vitro translation... - why not? (Jun/22/2006 )
so i see its expensive.... ....any other disadvantages??? it is much faster than all the E-coli mammalian etc....
if you'd want to do it which system you choose? thanx a lot!
I've used it and I find it very good. The kit I used was the TNT Quick from Promega.
When you purify proteins from E. coli, e.g. using GST tags, you have relatively pure proteins; if you want to use the proteins from reticulocytes as such, you should keep in mind that the extract will be full of proteins from the reticulocytes. The advantage is that you will have most of the modifications you have if you express the protein in mammalian cells (not the one made by nuclear proteins, that you are not supposed to have in a nucleus-free cell ).
You should check if your proteins have a T7 (or Sp6) promoter because it's required for the T7 (or Sp6) polymerase to transcribe the cDNA in mRNA.
further more you can make changes in the translation, for example use puromycin or tRNAs to incorporate special chemical groups. i used quiagen system, which also works nicely. but if you have a protein, that's easily expressed, it's still to expensive ....
these protocols are quite tough in sense they need several adjustments(salts conditions, purity of plasmid and even the fact of a linear / circular plasmid can helps from 10 to 100 folds )
RRL lysates are ok for in vitro translation, but modifications such as phopshorylations that serves in some experiments as a activation signal can occur.