HPLC for Protein Separation and Identification

Advanced Biotechnologies adbio at adbio.co.uk
Thu Jan 29 03:28:34 EST 1998


In article <34CF59A7.496C at rsgis4.tamu.edu>, yuanxiang Zhou <y-
zhou at RSGIS4.TAMU.EDU> writes
>Dear Netters:
>
>I am seeking for answers for a question.  Can HPLC or other
>chromatography-based methods be used for the purpose of separation and
>identification of proteins from the crude extracts of plant tissues?  We
>have introduced into tobacco a gene which encodes for a glycoprotein. 
>Even though we have not yet purified the protein from its
>native-occurring organism (very difficult to do so according to the
>literature), and there is currently no possibility for us to raise its
>antibody, we still want to try some altenative ways to have the job
>done.  The first thing we would like to know if HPLC can be used to
>separate and purify proteins, in the absence of a standard for
>comparison.  Does anyone have any valuable suggestions/recommendations? 
>You kind help is highly appreciated.
>
>Yuanxiang Zhou

Yes, it can. The main problem with HPLC is the high pressure, which can
denature proteins, and possibly clog up your column if the protein
extract is too viscous. Furthermore, you will need to make sure that
your solution has been filtered through a 0.22um filter, otherwise you
are pretty sure to increase the pressure in the system by clogging up
the column. I would suggest you start with a bulk precipitation
(ammonium sulphate), desalt, followed by an anion exchange column, (use
only aqueous solutions and elute using KCl), and finally fun down some
type of gel filtration column, tha elutes acording to size. That a
pretty good basis on which to start.


Don't use organic solvents
Use as large a width of column and as low a pressure as you can
Use low flow rates
Filter solution

You may also want to check the literature and see if you can find a
ligand-based dye, such as the MIMETIC types to selectively bind your
protein in an affinity-type mamnner. Also, is your protein any more heat
stable than the host proteins? Then simply heat it up a little (40-50°C)
and denature the contaminants, which you can then spin off.

Basically, yes, you can use HPLC. But remember, the drawbacks are why
Pharmacia introduced low and medium pressure systems, for exactly some
of the reasons I have given. Get back to me if you have any trouble.
Columns you can get from pretty much anywhere, but Whatman may do the
best ones.

Ariel Louwrier


--                                                       ______
Dr. Ariel Louwrier, Project Manager,    
Advanced Biotechnologies Ltd.,
Units B1-2, Longmead Business Centre,
Blenheim Road, Epsom
Surrey KT19 9QQ, UK.
Tel:  01372 723 456             E.mail:         ariel at adbio.co.uk
Fax:  01372 741 414             Web Site:       http://www.adbio.co.uk/




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