Hello Jacob,
I am not entirely sure why this has happened to you...
A couple of suggestions that may help:
1. dial down the TRIS to 20 mM - you can also use TRIS-HCl (unless the
second buffer is there for some other reason).
2. try a 'stronger' resin. I know in the past I've always strongly advocated
selectivity over binding strength (i.e. HIS-Select resin over Ni-NTA and its
many versions) but in this particular case there may be something weird
going on between HIS-Select and Calcium (such as gradual displacement of
bound ions, perhaps?). So in this case the use of His-Trap or Ni-NTA etc.
may be justified.
Let us know how it goes!
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Jacob
Keller
Sent: Monday, November 16, 2009 3:06 PM
To: [log in to unmask]
Subject: [ccp4bb] IMAC and Low Ionic Strength
Dear Crystallographers,
Recently I incubated a small amount of His-select resin with bacterial
lysate in
(50 mM TRIS-HEPES pH 8.0, 10 mM CaCl2),
and washed the resin, for certain experimental reasons, with
(50 mM TRIS-HEPES pH 8.0, 0.2 mM CaCl2),
at which point a significant amount of my protein fell off the column. The
ratio of my protein : background proteins was much higher in these washes,
implying that my protein probably had bound, but was now falling off the
column due to the 10mM==>0.2mM CaCl2 transition. Has anyone had problems
using such low ionic strength wash buffers? Is it possibly necessary for the
his-metal-ion interaction?
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [log in to unmask]
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