Dear Eric,

There are several essential variables that govern protein chromatography
(whether affinity, ion exchange, sizing, or other). It would be silly of me
to reproduce a protein chromatography handbook here, so instead I would just
list some practical pointers:

If your column is e.g. 10-15 ml of any HF agarose (High-flow, crosslinked)
packed into say an XK16 column (16 mm i.d.) then you can expect flow rates
anywhere between 9-14 ml/min with a low viscosity buffer. Naturally, things
tend to slow down if you have viscous buffers or suspended
microparticulates. Practically speaking, clarified E. coli lysate can
typically be loaded on such a column at 7-10 ml/min whereas clarified insect
cell lysate tends to require 5-8 ml/min flow. This of course assumes that
the back pressure is monitored and kept within reasonable limits (most
manufacturers give you considerably lower pressure limits than both the
column and the resin can actually withstand). Things to watch out for
include exponential clogging - if your flow rate is slightly too high, the
resin begins to compress, causing further increase in pressure, which in
turn compresses the resin even more and so on until either the upper limit
on pressure is triggered or the column is crushed/exploded. Obviously,
larger i.d. allows for higher flow rate - for instance, 50 ml HF agarose
packed into an XK26 column can usually sustain 20+ ml/min flow, likewise 150
ml of the same resin packed into an XK50 can do 40+ ml/min. Conversely, the
larger the column (assuming the same design and materials) the less pressure
it can withstand (e.g. ~1.8 MPa for XK16, ~1.2 MPa for XK26, and only ~0.6
MPa for XK50).

For resins made of polystyrene (such as e.g. MonoQ/S or Q15/S15, etc.)
crushing is less of an issue (the beads tend to be 'springy') whereas for
size exclusion columns made of e.g. Superdex or Sephacryl crushing can be
quite a problem. For somewhat exotic resins such as MonoBeads, ToyoPearl,
etc. the maximum allowable pressure can exceed that of the vessel they're in
(which is why stainless steel columns are sometimes employed with these
resins).

If you have to deal with particularly nasty lysates, don't forget that
batch-binding can be an easy and practical (but somewhat messy) solution. In
some cases I even had to resort to washing the resin on a Buchner funnel
with a glass wool filter. Anything to get the protein.

Good luck. Happy purifying!

Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Eric
Dollins
Sent: Thursday, February 28, 2008 5:26 PM
To: [log in to unmask]
Subject: [ccp4bb] Off topic: General rule for maximum flow rate for affinity
column?

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, [log in to unmask]