Bio-Rad Profinity IMAC Resins - Manual (Page 47)

43
Section 16
Troubleshooting Guide
Sample is too viscous High concentration of host
nucleic acids in lysate
Insufficient amount of
homogenization buffer
Viscosity of extract can be
reduced by addition of
Benzonase nuclease
(1.7 units/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 Reduce 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 Check expression level of
protein by estimating the
amount in the extract,
flow-through, eluted
fraction, and pellet upon
centrifugation. Use western
blotting with anti-6xHis
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 concen-
tration may bind to the
surface of the tube
Problem Possible Cause Solution
10001677B.qxd 1/28/2005 12:42 PM Page 46