Bio-Rad Nuvia™ IMAC Resin - Manual (Page 12)

8 Bio-Scale Mini Nuvia IMAC Ni-Charged
Section 6
Quick Solubility Screening Protocols
Before choosing a native or denaturing purification protocol, it is
useful to determine both the approximate expression level of a
protein and whether the overexpressed target protein partitions
into the soluble or insoluble fraction. Soluble proteins are typically
purified with the native purification procedure, while insoluble
proteins must be solubilized in stringent denaturants (urea or
guanidine) and are purified with the denaturing procedure.
The following procedure provides a quick screen for solubility and
expression level:
1. Pellet ~2 ml of E. coli culture by centrifugation at 4,000 x g for
10 min at 4°C.
2. Resuspend the pellet in 500 μl of PBS and sonicate on ice for
60 sec, in 10 sec pulses. Remove 50 μl of sonicate and label
as the Total sample. Centrifuge the lysate at 12,000 x g for
10 min at 4°C. Transfer the supernatant to a clean tube.
Remove 50 μl of the supernatant and label the tube Soluble.
3. Resuspend the insoluble pellet in 500 μl of 6 M urea in 1x PBS
and sonicate on ice for 60 sec, in 10 sec pulses. Centrifuge
the lysate at 12,000 x g for 10 min at 4°C. Remove 50 μl of the
supernatant and label the tube Insoluble.
4. To each of the 50 μl samples, add 150 μl of Laemmli buffer
and boil for 5 min at 95°C.
5. Load 10 μl of each sample on an SDS-PAGE gel.
6. Examine the soluble and insoluble fractions for the target
protein. Approximate the expression level and determine
partitioning of the target protein.