IMPACT kit problems

Jacqui Whiteway jwhitewa at chat.carleton.ca
Thu Apr 30 08:01:21 EST 1998


I'm using the IMPACT kit from NEB for purifiying my protein.  This kit
makes a triple fusion (protein of interest, intein, and affinity binding
protein) and relies on the intein to "release" the target protein upon the
addition of DTT.  

My problem is that my "purified protein" fraction contains contaminating
proteins.  4 bands (on an SDS-PAGE) appear to be forms of the intein
containing fusion construct (2 triples, 2 doubles), and one band is my
protein.  However, there is also another band around 30kD that I suspect
is B-lactamase (which, of course, is also overexpressed as it is the
marker on the vector).  

HAS ANYONE ELSE HAD THIS PROBLEM?

Thanks!


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Jacqui Whiteway

Email address: jwhitewa at chat.carleton.ca
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