On Thu, 2013-08-22 at 10:07 -0400, Bosch, Juergen wrote:
> Yes, I'm also surprised why people run gradients for the capturing
> step ?
Because we can. Joking aside, I've seen some examples where protein
eluted at relatively low imidazole and upon running the gradient there
remains some (minimal) overlap with non-specific binders. Mainly I just
never seen consensus on what is the right imidazole concentration for
the wash buffer (10mM? 50mM? may even depend on expession
circumstances), running the gradient takes that uncertainty away. Also,
batch method probably leaves more contamination behind.
One can run imidazole gradient in 1-2 hours, batch is not really much
faster, if at all. So if one has FPLC access, doing imidazole gradient
seems like a good standard policy. Benefits might be minor and rare,
but it is not much more work and certainly not worse than batch in any
respect.
My two cents (which in Russia turns into three rusty kopecks),
Ed.
--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs
