Yes,
However the even holier single method suitable for purification of
proteins from native sources is immuno-capture. If you have a good
antibody (i.e. an antibody that recognizes a native epitope) you can get
very high purity very quickly. If you don't have an antibody - you can get
polyclonals made very rapidly. Polyclonals are potentially even better for
immunocapture as some of them will recognize natively folded epitopes,
unlike monoclonals that may be raised against a peptide from deep inside
the protein.
Again, more details are needed :)
Artem
> The holy trinity of protein chromatography is ion exchange (IEX),
> hydrophobic interaction chromatography (HIC), and gel exclusion
> (GEC), assuming no affinity purification is possible. Depending on
> the abundance of protein, these three steps, perhaps in concert
> with additional chemical steps, including but not limited to salt
> precipitation and/or thermal denaturation, are often sufficient to
> purify proteins to homogeneity from natural sources. For
> minisculely abundant proteins, purification can be nightmarish.
> (Been there done that, got the dirty T-shirt.)
>
> The basis for any protein purification is an efficient assay for
> determining the presence of the protein in a mixture, even at low
> concentration. If you don't have a good assay (activity, antibody
> binding, spectroscopic ligand, etc., you are in dire straits indeed. But
> if you have an assay, you may not need to purify your protein at all to
> determine its oligomerization state in vivo. You may only need to pass a
> suitably concentrated crude extract containing your protein over a
> calibrated gel exclusion column. The emergence of the active fractions
> will provide an elution volume that will point you to the native MW, +/-
> 20% or so. The difference between a tetramer and an octamer (2X) should
> be cake using GEC. (Caveats apply: some proteins like to stick to GEC
> columns, but this can be suppressed with 100 mM NaCl, normally, but
> YMMV!)
>
> Cheers,
>
> --
> Roger S. Rowlett
> Professor
> Colgate University Presidential Scholar
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@mail.colgate.edu
>
> Jerry McCully wrote: .hmmessage P { margin:0px; padding:0px }
> body.hmmessage { font-size: 10pt; font-family:Verdana } Hi, All:
>
> Although it is off-topic, definitely I think I can get some help
> here because we crystallographers are dealing with protein
> purification almost every day.
>
> My protein was expressed in E.coli as a soluble octamer with or
> without his-tag revealed by gel-filtration. For the his-tagged
> protein, the final product is an octamer after Ni-column and
> gel-fil. However, purification of the non-tagged protein results in
> a tetramer because of a partially denatured condition.
>
> When I tested the enzymatic activity , it turns out the tetramer
> was active although both of them can be crystallized. Now I am
> wonderring whether its native form in human is an octamer or
> tetramer.
>
> I am planning to purify a little protein from human cells to verify
> its native form.
>
> Can anybody recommend some protocols?
>
> Thanks a million,
>
> Jerry McCully
>
>
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