"Nick Theodorakis" <nicholas_theodorakis at urmc.rochester.edu> wrote in
message news:052a85a2.612ffc32 at usw-ex0106-046.remarq.com...
> 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?
> >
Ion-exchange is a goodmethod of concentrating a protein. If it's pure, you
canelute it at a high concentration with a simple salt step, e.g., 500mM
NaCl.
>> 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.
>
Usually, the protein concentration is not a problem. I can think of the
following cases:
a) If your protein tends to precipitate at very high or very low
concentrations.
The latter case is more usual and the rsult is not limited to ion-excahnge.
b) A too high protein concentration might lead to irregularities during
loading or elution.
If your protein concentration is very high, it might affect the pH of your
buffer. Since your protein is also acting as a buffer, a high concentration
during loading or elution might shift the pH in the chromatographic buffers
used. The effect is esp. disturbing during elution. For example, in
anion-exchange the pH of the buffers has to be higher than the pI of the
protein, otherwise it would not bind. It is possible to elute proteins from
a column by a lowering of the pH ore by an increase in ionic strength. Let's
assume youre protien conc. is very high, you're running a salt gradient and
your protein is just about to elute. The protein will start eluting from the
top of the column. If the buffer-capacity of the eluted protein is very high
and the buffer capacity of your chromatographic buffer too low, you will
have a narrow zone of low pH running down the column. The lower pH will lead
to a dissociation of bound protein from the resin and you end up with a
broad peak and no separation.
However, I have worked with proteins as concentrated as 300mg/mL; it depends
on the specific protein, the buffer used for the chromatography, and the
buffer-capacity. My personal rule is, use buffers at 30mM or higher and not
more than 0.5 pH-units away from their respective pKa.
>> >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
The still do.
> to see if they still have them. Also, there used to be a book by
> Scopes, called something like, "Principles of Protein
> Purification. [snip]
>> 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.
[snip]
A very good book, esp. for beginners.
Cheers,
Achim
___________________________________________
Achim Recktenwald, Ph.D.
StressGen Biotechnologies, Corp.
Protein Chemistry
