Brett <Brett.Oconnell at students.vu.edu.au> wrote:
>I intend to separate some proteins by DEAE sephadex
>ion-excheange chromatography. My concern is this:
>>I have not found any theory regarding the relationship
>between the concentration of the sample to be applied
>to the column and the dimensions of the column.
>>My sample is quite dilute (~0.35 mg/ml total protein in
>about 9 ml). Can anyone give me some insight into the
>appropriate column size I should be using? Does the
>sample concentration matter?
>
To a first approximation, the total amount of protein is more
important than sample concentration for determining column size.
Indeed, some people have used ion-exchange to concenrate
proteins.
There is usually soem info on the binding cpapcity of the resin
you are using on the 'spec sheet.
>As an aside, I am wondering how many people actually
>read these bionet newsgroups since they tend to be quite
>empty. Is this because people read but don't post, or
>because the answers to the questions that are asked are
>too obscure for those who do look through bionet??
>I'm just concerned that I'm throwing my question out to
>only about 10 people or something.... So if anyone knows
>of a place I might be more likely to get a response, that
>information would also be much appreciated.
>
bionet.general is pretty quiet, but more useful groups are
bionet.molbio-methds-reagnts and bionet.proteins.
As for protein purification, Pharmacia used to sell (or even
sometimes give away) booklets on ion-exchange and gel filtration
chromatography that were quite useful. I don't if they still do
(and they are owned by Amersham now), but it might be worth a
call
to see if they still have them. Also, there used to be a book by
Scopes, called something like, "Principles of Protein
Purification." (I don't remember the exact name; people used to
just call it "Scopes")
Ah, here it is; I just looked on Amazon:
Protein Purification: Principles and Practice (Springer Advanced
Texts in Chemistry)
by Robert K. Scopes, Charles R. Cantor (Editor)
http://www.amazon.com/exec/obidos/ASIN/0387940723/qid%3D963321253/
103-6839053-4825458
It looks like it's still in press and even updated.
Nick
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu
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