Protein Purification Problem

Nick Theodorakis nicholas_theodorakis at
Wed Dec 13 12:02:02 EST 2000

In article <917mto$bar$1 at>,
  engelbert_buxbaum4 at wrote:


> > These fragments differ in size by only 20% and in pI by 0.23 units.
> There
> > are no affinity tags on the fragments.
> The fusion partner should have most of its original affinity left, so
> simply passing it over the same column that was used for purification of
>  the fusion protein might remove it, your protein of interest would be
> in the flow through and wash.
> > Using a starting material of 500ug/ml, should it be possible to
> separate
> > them by ion exchange (merits of anion vs cation??) ?? by
> size-exclusion
> > chromatography??
> pI and MW would not seem to be the most obvious separation parameters
> here (although chromatofocussing would probably work, it's just quite
> expensive).

You'd be surprised by what you can separate by ion-exchange if you are
careful. When my wife was in grad. school, she used to make chemical
derivatives of cytochrome c, and would separate out singly-modified
isomers (i.e., single modification, but on different lysines) with ion-

Reversed-phase might also work.


Nick Theodorakis
nicholas_theodorakis at

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