Ppt'ing reticulocyte lysate translated proteins

johns053 at MC.DUKE.EDU johns053 at MC.DUKE.EDU
Wed Jan 22 22:26:50 EST 1997


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     You need to degrade the aminoacyl-tRNA so that the 35-S 
     methionine-tRNA is not precipitated.  You can use the method from the 
     Promega manual, although I think more folks use a hot TCA protocol 
     where you add TCA to 10%, heat for 15-10 min at 90-100 degrees, then 
     isolate the precipitate on glass fiber filters.  For references, see 
     the cell-free translation system section of Methods in Enzymology, 
     vol. 101.
     Stewart P. Johnson


______________________________ Reply Separator _________________________________
Subject: Ppt'ing reticulocyte lysate translated proteins
Author:  NJH at mail.nerc-oxford.ac.uk at Internet
Date:    01/21/97 12:27 PM


Does anyone know of a simple method to precipitate and then count 35-S 
labelled proteins transcribed in rabbit reticulocyte lysate? I have tried 
regular TCA precipitation (spot on Whatman, 10% TCA, then washing) but the 
counts don't agree with the gel. There is a method in the Promega manual that 
involves treating with NaOH/H2O2, then precipitating with 25%TCA/2% casamino 
acids, followed by vacuum filtration on to glass filters. Does anyone know if 
this method is good?
     
Thanks.
     
Nigel


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Date: Tue, 21 Jan 1997 17:27:04 +0000
From: NJH at mail.nerc-oxford.ac.uk
Subject: Ppt'ing reticulocyte lysate translated proteins
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