Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 186)

Appendix F
Sample Preparation, Application, and Analysis Tips
Topic Tips and Recommendations
Sample
Preparation
Sample resuspension Resuspend harvested bacterial cells (wet or frozen) at a 1:10–1:20 (w:v)
and volumes
ratio of cell (g) to lysis buffer (ml). For example, resuspend 1 g of
cell in 10–20 ml of lysis buffer. Use a minimum of 10 ml of lysis
buffer for cell weights <1 g to facilitate cell lysis and to minimize
sample loss during preparation and handling.
Sample lysis — Use volumes 10 ml, even for small amounts of harvested cells.
sonication Adequately immerse the sonication horn to avoid frothing the cell
suspension. Select a sonication vessel that allows the tip of the
sonication horn to remain immersed during bursts.
Use short (30 sec) sonication bursts interspersed with frequent
cooling of the sample on ice. Excessive sample heating from
prolonged sonication times or high output settings can lead to
fragmentation of proteins and poor recovery of the protein of
interest.
Monitor the viscosity of cell suspension during sonication to
ensure adequate disruption of cell walls and nucleic acid fragments.
Highly viscous samples may benefit from pretreatment of the cell
suspension with DNase.
Sample lysis — Chemical lysis of bacterial cell pellets with buffers containing
chemical disruption detergents can result in decreased binding of GST fusion proteins
to the cartridge compared to samples prepared by mechanical
disruption (sonication). Yields can be 20–40% lower, depending
on the protein characteristics.
To minimize lysate viscosity, add a nuclease (such as benzonase)
to the resuspended pellet. Add benzonase at 25 U/ml (or DNase
at 100 U/ml), and incubate for 10 min at room temperature.
Sample clarification Clarify and filter the prepared cell lysate prior to application on the
Profinia system. Following cell disruption, centrifuge the cell
suspension for 15–30 min at 6,000 x g or greater. After obtaining
the soluble or insoluble fraction containing the protein of interest,
use a low-protein binding 0.45–1.0 µm filtration device to clear any
particulates not removed by centrifugation. For small sample
volumes (<50 ml), a 22–32 mm syringe filter is generally sufficient.
For larger sample volumes, it may be necessary to use a vacuum
or pressure-driven filtration device with sufficient surface area to
efficiently remove suspended particulates.
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