Elution from Glutath. beads

Dennis Templeton djt2 at po.cwru.edu
Thu Mar 16 14:25:32 EST 1995


In article <3k46j5$kjd at research-01.ski.mskcc.org>, Z-Suldan at ski.mskcc.org
(Zal Suldan) wrote:

> In article <1995Mar13.184105.2199 at news.unige.ch>
> ossipow at sc2a.unige.ch (ossipow) writes:
> 
> > I have troubles and irreproducibility when I try to elute GST fusion
> > proteins from glutathione sepharose beads (purchased from Pharmacia),
> > even with a new batch of reduced glutathione (therefore presumably 
> > not oxidized) that I use at 10 to 15 mM in the presence of 0.1% triton 
> > for 10 min. at 37c. Does anyone has a good trick ??
> 
> This is strange. We get great, reliable elution at 10minutes, Room
> Temp, 5.0mM glutathione, and no Triton. And the Sepharose is even
> better than agarose.
> 
> Have you checked the pH of the glutathione solution used to elute? I've
> found elution drops off significantly when pH is below 7.5 with best
> elution when pH = 8.0 - 9.0
> 
> Zal
> All opinions are mine and not of MSKCC

What Zal says is absolutely true. We did a survey here a while ago and pH
was the central important feature of successful elution. Remember that GSH
is acidic, so affects your elution buffer pH. We use 50 mM Tris pH 9.0 +
20 mM fresh glutathione. pH 7.5 doesnt work very well at all.

Dennis

Dennis Templeton
Institute of Pathology
CWRU School of Medicine
djt2 at po.cwru.edu



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