Hi Jacob,
an alternative explenation might be your protein does not bind via the
tag to the resin but unspecifically. What happens if you add the
recommended NaCl concentration? Does it fall off too?
Just a thought,
Jürgen
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
On Nov 16, 2009, at 16:05, Jacob Keller <j-
kell...@md.northwestern.edu> wrote:
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: j-kell...@northwestern.edu
*******************************************
