Protein purification

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Dec 19 23:59:19 EST 2001


> I would explore the active site of this protein, and use
> of proteases. Many proteases are opportunistic, attacking
> active sites of enzymes, etc, when they are exposed. You
> could consider adding binding partners, substrates, etc
> in the presence of proteases.

Post-IMAC soup of proteins is inherently dirty. Adding proteases will make
it worse, in most cases. Many proteases will eat off exposed flexible loops
and other things - again, more dirt and nonhomogeneity in the sample. Add
into the equation the fact that normally one adds protease inhibitors to
cell lysate, in order to prevent nonspecific proteolysis...

> Consider too, techniques like circular dichroism and
> dynamic light scatter. You say you are concerned about
> structural differences. I think these should be
> possible to identify by these techniques. You should
> be able to at least discern between a mixed and a
> pure population of folds.

Both CD and LS are nondiscriminatory. Especially if aggregation is present,
LS will give you a very uncertain result - regardless of what's really
happening in solution. CD will tell you what IS in solution i.e. the
available secondary structure, but it won't necessarily predict what it
can't see i.e. disordered, misfolded protein. These techniques are best
applied to pure samples.

There is no free lunch :(

A.G.E.





More information about the Proteins mailing list