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

"Khadka, Deepak NICHD khadkad at mail.nih.gov
Sun Jan 20 17:10:43 EST 2002


Yes, I was indeed talking about Maltose binding protein. Let me be clearer.
I have a MBP-Dlx5 fusion protein which was purified by using amylose resin.
My purpose was to use this fusion to 'pull down' other proteins that bind
with Dlx5 and purify the whole complex again by using amylose resin. My
problem was that those other proteins (Dlx3, Msx-1 and Dlxin-1 etc.) bound
with the resin even in absence of my fusion protein making it impossible to
use this system.

I eluted the resin bound materials by boiling the resin in Laemmli buffer.

I also tried eluting with the buffer containing 10 mM maltose. The elute did
not contain the test proteins as if the test proteins did not bind with
MBP-Dlx5 fusion. That is less likely because their interaction (I do not
know the efficiency) is known.

Deepak

> ----------
> From: 	Phil Harrison
> Sent: 	Friday, January 18, 2002 3:54 PM
> To: 	proteins at net.bio.net; Khadka, Deepak (NICHD)
> Subject: 	Re: Tricks to eliminate non-specific binding of proteins to
> amylose resin, any ?
> 
> 
> 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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