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
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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
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