Bio-Rad Nuvia™ IMAC Resin - Manual (Page 23)

Nuvia IMAC Ni-Charged Resin 19
5. Collect fractions from wash steps.
Pool recovered unbound proteins with fractions collected in
step 3.
6. Elute bound proteins with 5 column volumes of elution buffer.
Collect 2 ml fractions, or approximately 0.2 column volumes
each.
The choice of elution buffer will vary depending on the
procedure used. For example, a range of imidazole
concentrations (100–500 mM) may be used to elute bound
protein from the Nuvia IMAC resin.
7. Repeat elution steps 2 to 4 more times.
Save the eluates for further analysis (A
280
, SDS-PAGE, ELISA,
etc.).
IMAC Purification under Denaturing Conditions
In some cases it may be necessary to use denaturants such as
urea to solubilize inclusion bodies, which are not generally in their
native conformation. To perform this, up to 8 M urea (or 6 M
guanidine HCl) may be used in the binding, wash, and elution
buffers listed above. Elution is still achieved by increasing the
imidazole concentration.
Note: If using guanidine HCl (GuHCl), it must be removed from
purified samples prior to loading onto SDS-PAGE gels to prevent
precipitation. Proteins that have been lysed and adsorbed onto
the column with guanidine HCl may be washed and eluted with a
urea-based buffer.
Often the protein can be restored to its native form. To do this, the
denaturant used to lyse and purify the sample must be removed
using dilution, dialysis, or size exclusion chromatography.
Renaturation of the protein while it is still bound to the IMAC
column is a good alternative and offers several advantages.
Aggregation may be kept to a minimum if the protein refolds on the
column when the denaturant is removed. Higher concentrations of
the refolded protein may therefore be collected. Guidelines for
on-column renaturation are suggested on the following page.