Brett <Brett.Oconnell at students.vu.edu.au> wrote:
>I intend to separate some proteins by DEAE sephadex
>ion-excheange chromatography.
... snip..
If you're not confined to DEAE you should start IEX with something
like Q-sephadex. As it's a strong base, you won't have any trouble
with the buffering capacity of DEAE and will be able to use it over a
wider pH-range.
.. snip..
>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.
... snip..
It's called loading capacity and is listed either on the pamphlet
delivered with your chromatography media or on the Pharmacia home page
(the maker of sephadex column media).
... snip...
>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?
>See above.
>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....
I'd prefer 1 qualified answer than 100 stupid.... ;-)
...snip...
Dear Brett,
please excuse my directness, but you obviously should read up on the
theory and practice of IEX. The more you know about a method the
greater the advantage. IEX is a vary versitile method allowing high
degrees of purification - however - you must know what you're doing.
As much as I may often wish it to be, not type of chromatography is a
simple "apply sample, elute single cristall" method. There's no high
road to science. :-(
Harold
