postranslational analysis by TCA

Stephen R. Lasky Stephen_Lasky at brown.edu
Fri May 6 10:30:33 EST 1994


In article <CpD9xt.HI3 at umdnj.edu>, bawagan at umdnj.edu (Hinayana Bawagan)
wrote:

> 
>      I am going to use the rabbit reticulocyte lysate system to do the in
> vitro translation.  I want to solicit any information and if not too much to
> ask, a protocol on doing a TCA analysis to determine percent incorporation
> of radioactive label.  I have a protocol but I wish to have others as basis
> of comparison. Thank you very much.

Most published protocols work, try it and if it gives you linear
incorporation in a time course control experiment, use it.  

Also, from my experience in the late 70's it depends on what label you are
using. If you are using tritiated aa's, you need to bleach the filters with
H2O2.  If you are using 35S you don't need to bleach and can just ppt with
hot 10% TCA (to deacylate the amino acylated tRNA's), then a wash with cold
10% TCA and a couple of washes with cold 5% TCA. You can do this in a batch
(don't stir the filters directly or they will fall apart, you have to use a
double boiler type of setup). I think that we found that you need to use at
least 3 mls per filter for efficient washing.

hope this helps.

-- 
***************************************************************
Stephen R. Lasky, Ph.D.       Brown University/Roger Williams Medical
Center
e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
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A nuclear war could ruin your whole day.
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