Bio-Rad Nuvia™ IMAC Resin - Manual (Page 19)

Bio-Scale Mini Nuvia IMAC Ni-Charged 15
Section 11
Troubleshooting Guide
Cartridge clogging or slow flow rate
Particulates in samples
Filter all samples and 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 in eluent
Low level of target
Check expression level of protein in
starting SDS-PAGE material
Target protein not binding Check levels of target protein in lysate,
flowthrough, wash fractions, and eluted
fractions
Check for presence of histidine tag with
antihistidine antibody
Target protein in flowthrough
Histidine tag not
accessible
Purify protein under denaturing
conditions to expose histidine tag
Reclone histidine tag onto opposite
terminus (N- or C-terminus)
Proteolysis and removal Include protease inhibitors in histidine-tag
lysis buffer or purify in the cold
Precipitation during purification
Binding capacity of
cartridge exceeded
Load less sample
Protein aggregating Include low levels of a detergent
(0.1% Triton X-100, Tween 20)
Include glycerol up to 10%
Protein too concentrated
in step elution
Elute with imidazole gradient
Eluted protein is impure
Contaminants co-eluting
Elute with imidazole gradient
(10–500 mM) rather than step elution
Increase imidazole in the wash to increase
wash stringency, but keep below 40 mM
Target protein is degraded
Proteolysis of target
Add protease inhibitors to protein lysate
Purify at 4°C or under denaturing
conditions