Re: [ccp4bb] how to purify protein in its native form

[Roger Rowlett]

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: 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 Rediscover Hotmail®: Now available on your iPhone or BlackBerry Check it out.