Bio-Rad Profinity IMAC Resins - Manual (Page 28)

Section 11
Troubleshooting Guide
Problem Possible Cause Solution
Cartridge clogging Particulates in samples Filter all samples and buffers
or slow flow rate or buffers through 0.2 µM filter prior to
application
Sample too viscous Add nuclease to lysate to
degrade DNA. Centrifuge
and filter lysate again
No target protein Low level of target Check expression level by
in eluant protein in starting SDS-PAGE
material
Target protein not Check levels of target protein
binding, or eluting in in lysate, flow-through, wash,
wash fractions and eluted fractions. Check
for presence of His-tag with
anti-His antibody
Target protein in His-tag not accessible Reclone His-tag onto opposite
flow through terminus (N or C-terminus)
His-tag not accessible Purify protein under denatur-
ing conditions to expose His-
tag
Proteolysis & removal Include protease inhibitors in
of tag lysis buffer (or reaction), or
purify in the cold
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