purifying translation products

Technical Assistance TechAssist at pierce.geis.com
Tue Sep 17 08:42:44 EST 1996


Mara Casar wrote:
> 
> Hi-
> 
> I'm trying to do an in vitro translation using BMB's biotinylated
> lysine-tRNA reagents (instead of 35-S labeling).  When I do the
> translation in reticulocyte lysate and run an aliquot on a gel,
> transfer to nitrocellulose, and then detect with streptavidin-HRP and
> ECL, I get a lot of other bands lighting up.  Retic. lysate supposedly
> has only a few endogenous biotinylated proteins, though.  Has anyone
> out there in Internet-land (a) used biotinylated lysine in in vitro
> translations or (b) had a similar problem and can offer advice?  I'm
> not able to see any difference between my control translations without
> RNA and any of my samples.
> 
> Thanks in advance!
> 
> -Mara Casar
> Department of Biochemistry
> Duke University


What you may be experiencing is non-specific binding of Streptavidin to cell 
surface proteins. Streptavidin has an amino acid sequence in its structure very 
similiar to that of fibronectin. This can cause problems of non-specific binding in 
cell lysates. You may wish to try Neutravidin as a replacement for the Strep. 
Neutravidn does not contain the cell recognition sequence, and it has a lower pI. 
If non-specific binding is the problem, Neutravidin wil eliminate it. If the problem 
is other biotinylated proteins, then the Neutravidin will not help.

Tom Brotcke/Pierce TA



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