1. Using a low pressure system (glass, plastic, no metal),
2. Using a strong exchanger (like QAE-Sephadex)
3. Using a concentration gradient of a volatile buffer (NH4OAc,
PyOAc, NH4HCO3) up to high concebtrations (2M buffer or higher).
We are routinely purifying multigram quantities of raw peptides
(though, synthesized in solution) on SP-Sephadex and often are
obtaining products with 95%+ purity. Well, they are all basic.
If that fails, you can try a well-forgotten method of counter-current
distribution (if you can get access to a device). An alternative to
this is even more forgotten partition chromatography on Sephadex G-25
(you can read about it in Houben-Weil Bd.15 Pt.2)
On 01/12/1999, Jenny Shipway wrote:
> Hello everyone, thanks for all your (very quick!) replies.
> i) The reason I'm not using RP is that I already tried that, and couldn't
> resolve the peaks. I thought IE would be easy in comparison!
> ii) My peptide *is* N-capped, so cation IE would be out, even if I didn't
> now have far too much fear of IE columns to consider ever buying another
> one.
> iii) The column I'm using is a brand-spanking-new Vydac VHP (DEAE) 5.00mm
> ID column. I'm told these shouldn't really have any metal atoms lying
> around, because of the type of matrix (spherical PS-DVB beads). I doubt
> it'd be bad enough to chelate everything I threw at it, anyway(?)
> iv) I'll give it a go with the 10% acetonitrile at pH2, like Andy Alpert
> has suggested, but I still think there's something really odd here that
> probably needs to be sorted out...
> NEWSFLASH!
> Since mailing the list, I've managed to get a peak! Sadly, it's not (I
> believe) the peptide I want: I shoved a set of pI calibration peptides
> down the column and eluted at pH8, 0-0.5M NaCl. This set included 6
> proteins with pI more than one pH unit lower than pH8. I got *one* peak!
> Is it just me, or this this not normal? In case anyone cares, they were:
> Amyloglucosidase pI 3.50
> Soybean trypsin inhibitor pI 3.75
> Beta-lactoglobulin A pI 5.20
> Bovine carbonic anhydrase B pI 5.85
> Human carbonic anhydrase B pI 6.55
> Horse myoglobin - acidic pI 6.85
> There were also 5 others with higher pI's.
> All the hardware I'm using is exactly the same as we use for RP (which
> works fine, including with my peptide), except for the column itself and
> two short sections of tubing.
> Any suggestions as to what I could try next? The people who sold it to me
> seem to be as confused as I am about what could be going on.
> Jenny
> PS: Is it normal to have to wait ages for these things to equilibrate? The
> signal at 210 seems to just go down and down for ages.
Vladimir Titov
Bokiron Ltd., Moscow, Russia
vmtitov@aha.ru