Rabbit Reticulocyte Lysate System

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Guy Sovak
Guy Sovak's picture
Rabbit Reticulocyte Lysate System

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

Fraser Moss
Fraser Moss's picture
I have used it for on vitro

I have used it for on vitro protein synthesis and for the incorporation of unnatural amino acids in to in vitro translated proteins.

Guy Sovak
Guy Sovak's picture
Hi Frasermoss,

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

Fraser Moss
Fraser Moss's picture
what are you rescuing?

what are you rescuing? protein folding? translation?

Why will the rabbit reticular lysate system be suitable for your experiment?

Guy Sovak
Guy Sovak's picture
Refolding of a protein.

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

Fraser Moss
Fraser Moss's picture
I don't think there are any

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
Fizz's picture
Actually there is a secret ;)

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.