In general, I would wherever possible bind the protein in batch mode
- 15-30 mins with occasional swirling.
And then pour in a column, wash and elute. If washing and eluting is
very slow, I would apply a little pressure that one time, but the
next time make sure to choose a wider column.
Another common "mistake" is to take too much resin - the minimal
amount to bind the protein should be used, any more will usually lead
to more impurities upon elution.
Mark J. van Raaij
Dpto de Bioquímica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
http://web.usc.es/~vanraaij/
On 28 Feb 2008, at 23:26, Eric Dollins wrote:
Dear protein purifiers,
Off topic question: Is there a general rule for how fast you can load,
wash and elute from affinity columns, e.g. glutathione agarose? The
product insert from Sigma says load under gravity flow. For the
volume of cell lysate I have, gravity loading would take an
excruciatingly long time. I want to hook up a peristaltic pump to
speed things along, but don't really have a feel for just how fast one
can load a column in general (I realize this is also dependent on the
construct, the buffer, etc). What about the subsequently wash or
elution?
Thanks for help
Eric
--
D. Eric Dollins, Ph.D.
C266 LSRC, Research Dr.
Duke University Medical Center
Durham, NC 27710
(919) 681-1668, [EMAIL PROTECTED]
