chelex -100 column

Don Demuth demuth at biochem.dental.upenn.edu
Thu May 26 11:28:37 EST 1994


In article <2rrh2k$qtj at bengal.oxy.edu>, akopian at cub.math.oxy.edu (Gabriel Akopian) writes:

|> 	Recently we ran calmodulin through a chelex column without  
|> buffering the protein or the column...We're having two problems; (1) is  
|> the column pH is at about 10-11 and is practically impossible to  
|> lower...so our protein is in a very basic medium, and (2) we're having  
|> trouble removing calcium from the calmodulin.  With each subsequent pass  
|> through the column, we're lowering the calcium concentration but it is  
|> taking several passes to accomplish this...Can anyone give any advice or  
|> comments.


	We have also used Chelex resin to strip calcium from proteins but do it a
bit differently than you describe.  First, the samples are suspended in a MOPS
buffer, ph 7.5.  Is there a reason that you are not buffering your calmodulin? 
Second, we usually strip batchwise as opposed to running the protein sample on a
column and as you have noted, it takes several passes (or batches) of Chelex to
completely strip the sample.  We routinely monitor the effectiveness of the
stripping reaction by adding a small amount of stripped protein to a solution of
fura2.  A sample is free of calcium when the fluorescence of fura2 in the
presence of protein does not differ significantly from its fluorescence in
excess EGTA.  Be glad to send you the details of our procedure if you're
interested.  Send an email message. 


			Don Demuth

_________________________________________________________________________________
demuth at biochem.dental.upenn.edu



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