MBP fusion protein does not bind to amylose?

[ZhongLingChai]

Dear all,

I have a fusion protein (not too big, about 75kd including the MBP)
that does not seem to bind to the amylose resins. I know this has something
to do with the particular fusion protein because another protein slightly
smaller is purified nicely with the same amylose. All the lysis/binding and
washing were carried out with column buffer recommended by NEB (Tris pH7.6,
NaCl and EDTA), and eluted by 10mM maltose in the same column buffer. I
thought about another possibility that the fusion protein had been bound,
but not been eluted. Hence I tried to elute with 100mM maltose. Nothing
happened. I wonder if this has something to do with the protein folding
that may hide the MBP binding domain. If so, does addition of 5mM DTT or
2-ME to the column buffer help? All the suggestions and comments are always
appreciated.

Regards

ZhongLin Chai
Ph.D. (Molecular Biology)
M. Agr. (Veterinary Virology & Immunology)
B. Agr. (Veterinary Sci.)
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Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital, Commercial Rd,
Prahran, Victoria, AUSTRALIA 3181
Telephone: (61 3) 9276 2698 (lab)
(61 3) 9276 2696 (office)
Fax: (61 3) 9529 6484
email: zhongl...@med.monash.edu.au
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