Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 187)

Topic Tips and Recommendations
Sample Screening — Before choosing a native or denaturing purification protocol,
Solubility Profile
approximate expression level and determine if the overexpressed
target protein partitions into the soluble or insoluble fractions.
Soluble proteins are typically purified with a native purification
procedure, while insoluble proteins must be solublized in stringent
denaturants (up to 8 M urea) and are purified with the denaturing
procedure. To determine the solubility profile:
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 for 60 sec
on ice, in 10 sec pulses. Remove 50 µl of the sonicate and
label as the “total” sample. Centrifuge the lysate at 12,000 x g
for 20 min at 4°C. Transfer the supernatant to a clean tube.
Remove 50 µl of the supernatant, and label as the “soluble”
sample.
3. Resuspend the insoluble pellet in 500 µl of 6 M urea in 1x
PBS, and sonicate for 60 sec on ice, in 10 sec pulses.
Centrifuge the lysate at 12,000 x g for 20 min at 4°C. Remove
50 µl of the supernatant, and label “insoluble” sample.
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.
Examine the soluble and insoluble fractions for the target protein.
Approximate the expression level and determine the portioning of
the target protein.
Sample Degradation Once cell lysis has begun, work quickly to get the purification
started. The longer a complex lysate sits, the greater the chance it
will degrade. Performing lysis at 4°C can also help minimize
degradation.
The GST lysis/wash buffer contains 5 mM EDTA and is an
effective inhibitor of metalloproteases. However, additional
protease inhibitors or the addition of a protease inhibitor cocktail to
the sample immediately prior to the cell lysis step may improve
the yield of target protein. Minimize the time between cell lysis
and application of the cleared lysate to the column. Keep the
sample on ice.
General Sample Keep cell suspensions on ice to avoid precipitation and
Preparation Tips degradation of the proteins of interest.
Freeze harvested bacterial cells at –20°C or below. Prepare cell
suspensions fresh and avoid freeze-thawing the cells or the
prepared lysate.
Keep samples of each fractionation step used during sample
preparation for analysis by SDS-PAGE or similar in order to
monitor the efficiency of lysis and recovery of the protein of
interest. For SDS-PAGE samples, preparing a 1:10 dilution of the
sample in sample loading buffer and loading 5–10 µl of the
preparation is generally sufficient for detection of the bands of
interest.
180