Small peptide purification blues

John E. Fox altabios at bham.ac.uk
Fri May 27 04:49:54 EST 1994


In article <01HCSQ4FBJAQ003YA5 at GW.AGR.CA>, RAMPITSCH at BCRSSU.AGR.CA says:
>
>I have a peptide query:
>
>        I'm trying to isolate a peptide about 1000 to 1500 Da (best guess)
>and probably cyclic. Initial purification steps are: acid precipitation
>(very inefficient) followed by ion exchange chromatography (which
>works well). I want to make polyclonals against the peptide and need
>more purification steps so I've set up a gel filtration column. Now I
>need to concentrate the sample before loading the column. The sample is
>about 20 ml in volume and contains approx 0.4M NaCl and 10 mM Tris buffer
>pH 7.5. The peptide is reasonably easy to produce, so we can tolerate 
>some loss.
>        
>        We're considering trying: 
>
>1) Ultrafiltration (but can't find any appropriate columns)
>2) Ammonium sulphate precipitation (the peptide appears to be quite soluble)
>3) Phenol (will the peptide go into the phenol? and how do we get it back?)
>4) ?????
>
>Thanks for any suggestions or protocols...
>
>Jeff 

How about trying the following:-
1. Initial clean up by ultrafiltration instead of acid precipitation. 
This should give better recovery.
2.  Purify by HPLC on a reverse phase column e.g. Vydac using
gradient of water/MeCN with 0.05% TFA.
3. This should give peptide without any salts.
4. If you want to keep the ion exchange, try removing salts with
a small column of Sephadex G10. 

You could also concentrate on a Sep-Pak column.



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