Trouble removing contaminating proteins from amylose resin

John Everett kellere at hotmail.com
Sat Feb 16 02:31:49 EST 2002


  I have created an amino terminal Maltose Binding Protein (MBP) 
fusion of my pet protein. Unfortunately...  when I bind the fusion 
to an amylose resin, three other proteins invariably bind as well.  
I have shown that the three hitch hikers are binding to my protein 
and not to the resin nor to the MBP.
I have tried varies amounts of NaCl ( 0 - 1M ), varying pH levels 
(5 - 8). I just can not shake the contaminants off. The contaminants 
stick to the fusion when the fusion is passed through ion exchange 
columns and size exclusions columns.

  I normally wash the resin with 20 column volumes of:
  20  mM Tris-HCL  pH 8.0
  200 mM NaCl
  10  mM BME

 If any one can offer a wash protocol that may help knock
 off the contaminating proteins, I would be very appreciative.

  Thanks in advance,
        John Everett

UMDNJ | Rutgers University
kellere at hotmail.com

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