Bio-Rad Nuvia™ IMAC Resin - Manual (Page 35)

Nuvia IMAC Ni-Charged Resin 31
Section 11
Troubleshooting Guide
Problem Possible Cause Solution
Sample is too viscous High concentration
of host nucleic acids
in lysate
Insufficient amount of
homogenization
buffer
Viscosity of extract can be reduced by
adding Benzonase nuclease (1.7 U/ml)
with 1 mM MgCl
2
to fragment bacterial
DNA. Incubate on ice for 15 min
Dilute sample by adding more
homogenization buffer
Sample application
causes column
to clog Insufficient
clarification of sample Prevent cell debris from clogging the
column by increasing the centrifugation
speed or filtering the sample
No protein is eluted Expression of target
protein in extract is
very low and is not
found in the eluate
Target protein is
found in inclusion
bodies or possible
insufficient lysis
Target protein
is found in the
flowthrough
Check expression level of protein by
estimating the amount in the extract,
flowthrough, eluted fraction, and pellet
upon centrifugation. Use western blotting
with anti-6x histidine antibodies, target
protein-specific antibodies, ELISA, or
enzyme activity determination
Apply larger sample volume
Minimize contact with hydrophobic
surfaces (that is, polystyrene tubes).
Proteins at low concentration may bind to
the surface of the tube
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 3, and 7)
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
histidine-tagged sequence placed at the
opposite terminus