HELP!!!!!with protein renaturation (in a soluble form).

Mike Faughn mrfaughn at unity.ncsu.edu
Tue Jun 11 09:44:02 EST 1996


Hello,

	The situation is this--  I would like to isolate plant proteins that
interact with my His-tagged protein (of viral origin).  I am
inoculating plants with the virus (which carries the His-tagged)
protein and am hoping to use a NiNTA column to purify protein
complexes.  Obviously I would like to do this under very gentle,
native conditions.
THE PROBLEM AT HAND--  Before spending tons of time trying to work out
the native binding conditions for my His-tagged protein derived from
infected plants I would like to "practice" with bacterially expressed
protein.  I have expressed and purified the protein using the pET
vector.  I have been using 8M urea and then slowly renaturing by
dialysis.  The problem is that all the protein precipitates by the
time I get down to a 200mM NaCl buffer.  This floculent stuff is no
good for column work.  
SO-- any ideas on how to get some soluble protein to "practice" with?

Thanksalot,
mike 




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