Scientist Solutions - Rabbit Reticulocyte Lysate System

gsovak Posted 12/20/2006 11:01:45 PM	Hi all, Did Someone used the Rabbit Reticulocyte Lysate System from Promega? I want to start doing in vitro Ubiquitination assay and add this lysate. I read several articles but would love to hear from anyone who used it. Guy
frasermoss Posted 1/6/2007 1:00:29 AM	I have used it for on vitro protein synthesis and for the incorporation of unnatural amino acids in to in vitro translated proteins.
gsovak Posted 4/12/2007 7:42:28 PM	Hi Frasermoss, The reticulocyte lysate assay that I wanted to do some time ago is comming once again to my bench :-9 So If you could tell me some tips that you have for me I would be happy. My Idea is to use a plasma membrane protein WT and Mutant and try to check if addition of chapeornes will change the rescuing of the protein. Guy
frasermoss Posted 4/12/2007 8:41:15 PM	what are you rescuing? protein folding? translation? Why will the rabbit reticular lysate system be suitable for your experiment?
guy Posted 4/15/2007 3:12:18 AM	Refolding of a protein. I want to try this in vitro system as I can add different chaperones and check their importance in the refolding of the protein of intrest. GUy
frasermoss Posted 4/16/2007 4:01:35 PM	I don't think there are any dark secrets to this system. I generally followed the manufacturer translation protocol and scaled it as appropriate for my application. One thing I did do was include 1 ul RNase inhibitor (Roche) to each reaction. But that was mainly to protect the tRNA+unnatural amino acid that I was introducing into the system. You will have to experiment with how much protein to run on blots if that is what you plan to do. It makes a lot of protein.
Fizz Posted 5/10/2007 10:23:54 PM	Actually there is a secret ;) The lysate is ruined after 2 freeze-thaw cycles. Calculate the reaction volumes you need and then aliquot it the FIRST time you thaw it. It can be stored at -80C for over a year however long term storage was in LN2. Apart from that, the other thing of some importance is that the protein amount produced depends a lot on the template. Linearized templates seem to generate higher amounts of protein. Another obvious detail (though often ignored) is that the salt type and concentrations seem to play a very important role. So you will have to do some experiments to get the system optimized. There are good details given in the Promega Protocols volumes as well.