Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 19)

Leftover Buffer After Ten Runs
• Each kit contains sufficient reagents for ten purification runs (equivalent
to 10 ml of resin). Depending upon which method is used (standard
vs. extended wash, rerun of same method, insertion of new cartridge
vs. using the same cartridge, etc.), some bottles may not be completely
depleted, while others may be nearly empty. To minimize the risk of
contamination, it is recommended to discard any unused buffers
rather than pool multiple old lots of buffer to refill bottles
Viscous Lysates
• The lysis buffers that are supplied in each kit are optimized for lysis by
sonication. A ratio of 10 parts 1x buffer to 1 part cell pellet (v/w) is
ideal for lysing cell pellets. Depending upon the density of the culture,
the power of the sonicator, and the condition of the sonication tip, the
final viscosity of the lysate may vary. Extremely viscous lysates create
system backpressure and run the risk of slowing down the flow during
sample loads
• To decrease viscosity, a nuclease should be added prior to sonication.
Benzonase (Novagen) or DNase are commonly used nucleases
Large Sample Volumes
• When low protein expression requires large culture volumes (>2 L of
starting culture), the lysate loading volume can exceed 45 ml, which
exceeds the volume that can be used in a standard sample tube. In
these instances, bacterial pellets can be lysed at 5:1 (v/w) ratios.
When concentrating starting samples at 5:1 ratios, it is strongly
recommended to add a nuclease to minimize sample viscosity
Section 8
Legal Notices
Purification and preparation of fusion proteins and affinity peptides
comprising at least two adjacent histidine residues may require a license
under US patent 5,284,933 and US patent 5,310,663, including foreign
patents (assignee Hoffman-LaRoche).
Expression and purification of GST fusion proteins may require a
license under US patent 5,654,176 (assignee Chemicon International).
Benzonase is a trademark of Merck KGaA.
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