Tricks to eliminate non-specific binding of proteins to amylose resin, any ?

Phil Harrison arsphys at cc.usu.edu
Fri Jan 18 15:54:35 EST 2002


I am assuming the MBP is maltose binding protein.  If I am in error, you 
can probably ignore this whole message,  but if it is maltose binding 
protein, why do you refer to the binding as "non-specific binding" since 
amylose is composed of glucose units, and when digested by amylase produces 
maltose.  It seems this is very close to the natural substrate.

My suggestion is to elute with a high concentration of maltose, say 100-500 
mM to occupy the binding sites on the MBP.  I'm not familiar with MBP, but 
are there any other factors that influence binding of maltose?  If metals 
are involved in the binding, elute with higher concentrations of EDTA, etc.

Good Luck,

Phil


At 04:32 PM 01/17/2002 +0000, you wrote:


>I am performing binding assay of some test proteins with MBP-fusion protein.
>When I try to purify the mixture by using amylose resin, most of the test
>proteins gets stuck to the resin. My buffer (both for binding and washing)
>is 50 mM PIPES (pH 7.0), 100 mM NaCl and 1 mM EDTA. Playing with the salt
>concentration and adding BSA did not help. Any suggestions ?
>
>Deepak
>NICHD/NIH
>
>---

Phil Harrison

USDA-Agricultural Research Service,
Forage and Range Research Lab
Utah State University,  UMC 6300
Logan, UT 84322-6300

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