help with protein purification

Gene Cutler genecutl at SP1.BERKELEY.EDU
Wed Dec 15 13:39:31 EST 1993



----------------------------Original message----------------------------
In article <9312101735.AA16550 at net.bio.net>,
J. Zwiazek <JZWIAZEK at VM.UCS.UALBERTA.CA> wrote:
>From: J. Zwiazek
>We are trying to purify an acidic protein from plant cells using DEAE Sephadex
>and Resource Q (almost identical to Mono Q) HPLC columns.In both cases we are g
>etting a nice separation of proteins except for the protein that we want to iso
>late which is present in most of the fractions, even very early ones. We tried
>a whole range of buffers of different pH, buffer and NaCl molarity, flow rates,
> etc. but nothing helps. We are quite puzzled because a similar protein is rout
>inely isolated from animal tissues using Mono Q and DEAE Sephadex columns.
>Has anyone experienced a similar problem? Any comments and advice would be appr
>eciated.
>Thanks,
>Janusz Zwiazek
>

One factor which leads to the presence of a protein in many fractions is
overloading, but that doesn't sound like the problem that you're having.
Perhaps your protein is forming protein-protein complexes with something else
or itself and this is affecting the separation.  Have you tried denaturing
columns?  Also, have you tried size exclusion chromatorgraphy?  If your
protein comes out all over the place from that column, it would indicate
some kind of aggregation or complex formation.  Also, if you increased
the amount of DTT in your buffers it might make a difference.




--gc
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   These possibilities are all logical, but I think you should first
make sure that the protein you have in almost all the peaks is really
YOUR protein. (maybe your identification method is not so specific).

Luck,
Wagner Fontes



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