Protein Purification Help

Jim Campanella jjc3 at LEHIGH.EDU
Mon Dec 9 12:10:49 EST 1996


I have a slight problem that may be fairly
simple to solve, but it's not obvious
to me at the moment-- probably because I
admit I'm not much of a biochemist. Perhaps
someone out there could help.

The problem is this. In a class I have been
teaching, we have been purifying an enzyme
as one of the projects. In the past, the
last purification step is a desalting step
using dialysis. The enzyme is dissolved in
the following common buffer:

20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA,
0.5% Tween20, 0.5% NP-40, and 0.5 mM PMSF.

The dissolved enzyme is then dialyzed 
overnight against the following buffer:

20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM
EDTA, 50% glycerol, 0.5% NP-40, 0.5%
Tween20, and 1mM DTT.

This works well to get a clean enzyme that
has activity, but I thought it might be
nice to modernize and speed up the
protocol for the class. So, I thought
a desalting column (eg. Bio-Gel P-6DG)
might be a nice replacement for the
dialyzing step.

My problems: 

A) I forgot that the dialyzing
step serves the secondary purpose of
getting glycerol into the buffer with 
the enzyme. So how do I get the glycerol
into the buffer if I use the desalting 

B) It seems to me that if I use the
column I'll be losing some salts 
that will be needed to help buffer
the enzyme-- salts that are still
present but equilibrated with dialysis.
Again, how do I solve the problem of
this loss?

It seems to me that I may be creating
more problems that I am solving by
trying to "simplify" this protocol.

Any suggestions would be greatly


Jim Campanella
Dept. of Biological Sciences
Lehigh University
Bethlehem, PA USA

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