I am facing problem in setting up in vitro translation reaction. It looks like I need an energy generating system like phosphoenol pyruvate and pyruvate kinase . There are kits available (NEB) also, but they are pretty costly. If I prepare cell extract and then use it for IVT will there be any problem? How much does omitting energy generating system affects the reaction? I have came across protocols for IVT but all uses the PEP and pyruvate kinase, is there any way to bypass it by using excess ATP?
Thanks in advance
In vitro translation should work, and it usually does, without adding energy generating systems. Try using an excess of ATP (3mM at least).
but from one paper "Prolonging Cell-Free Protein Synthesis with a Novel ATP Regeneration System" I came to know the real need of the energy regerating system i.e. to prevent the interaction between free magnesium and released phostphate. As I do not have those chemicals and enzymes, can I add Magnesium in excess and carry out translation? Or do you have any other suggestions?
No, mg in excess is likely to inhibit translation. Actually, it all depends on what system you are using, which efficiency you desire and what is the purpose of your experiment. Adding PEP and kinase of course gives optimal results, but if cost is an issue you can dispense of them for most uses. Just try...
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