Purification of proteins in denaturants

michael bausher mbausher at MAGICNET.NET
Tue Sep 17 14:27:13 EST 1996


Hello:

Your discription of the problem was brief, but you can use free
solution IEF at very high Urea concentrations (8 M for the Rotofor),
 but you have to increase the running temp to 15 C.  You also should
deionize the Urea .   The highest concentration I have used is 4 M. 
  You also have to deal with the ampholytes  later.    


Hope this helps,

Michael

michael bausher
USDA-ARS
Orlando
mbausher at magicnet.net


     From: zaphod at mcimail.com
     Date: 9/16/96  5:05:22PM
     To: protein-analysis
     Subject: Purification of proteins in denaturants
     
     Does any one have any good suggestions for purifying proteins in
     denaturants (e.g.,6M urea, 4M guanidine)?  I have a his-tagged
     protein
     which I've been able purify to a great extent by Ni affinity,
     but I'd like
     to have another chromatographic step.  The protein is only
     soluble in 6M
     urea or 4M guanidine.
     Sizing columns work poorly in these buffers and we have not
     found an
     ion-exhange resin to which the protein will bind in these
     buffers, either.
     Thank you.
     Blake Middleton        Internet:  zaphod at mcimail.com
     MCI ID # 465-2423        Bitnet: 
     zaphod%mcimail.com at cunyvm.cuny.edu
     
       "Ah, this is obviously some strange usage of the word `safe'
     that I
       wasn't previously aware of."                    --Arthur Dent
     
     
     





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