Hi Ed,
well if we hit the timer after lysis, say via cell disruptor then I have my
eluted protein in less than 1 hour, including 40 minutes batch binding. If I'm
pressed for time I bind only 20 minutes and then can have the protein ready for
the next step within 30 minutes after lysis. Some of you are then still
centrifuging and have not even started to load the column. [Quick jump to last
paragraph for the reason]
I see the disadvantage more in occupying an Akta for something that could be
easily done without one. Also a pump with gradient mixer may be an alternative.
But every protein is different and you should obey to your protein and not
stick to what you think is best, trial and error and hopefully moving fast to
some conclusions on how to best purify your protein.
What I noticed over the years is that frequently if the initial capturing step
takes too long you will end up with protein that is not necessarily as
functional as you wanted it. You can add cOmplete tablets (if you don't work on
proteases) of course, but sometimes you can't do that and then it gets
important to clean up the sample ASAP.
I had my protein once almost completely disappear because I thought I could go
quickly shopping while the centrifuge is spinning down the lysate. So I added
an extra hour instead of 30 minutes. Well that purification ended in nothing. I
believe I learnt something - did I ?
Just a thought
Jürgen
On Aug 22, 2013, at 1:47 PM, Ed Pozharski wrote:
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
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://lupo.jhsph.edu
