Bio-Rad Profinity IMAC Resins - Manual (Page 48)

44
No protein is eluted
(continued from
previous page)
Target protein is found in
inclusion bodies or
possible insufficient lysis
Target protein is found in
the flow-through
Increase intensity/duration
of disruption and
homogenization
If protein is insoluble, use
6 M guanidine HCl or 8 M
urea to lyse denatured
proteins (see Sections 8,
11, and 13)
Reduce imidazole
concentration in sample,
binding, and wash buffers.
An imidazole gradient may
be used to determine
optimal amounts for wash
and elution conditions
Check pH levels of
sample. A decrease in pH
may result during the
homogenization step or
during growth of the
culture medium. Adjust pH
to 7–8
The histidine tag may not
be accessible. Use
denaturing conditions to
purify protein or reclone
the plasmid construct with
the His-tagged sequence
placed at the opposite
terminus
Proteolytic cleavage during
fermentation or purification
has caused the histidine
tag to be removed. Add
protease inhibitors or
make a new construct with
His-tag attached to other
terminus
Problem Possible Cause Solution
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