Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 24)

2. Dialysis - Probably the simplest method for ampholyte removal is dialysis.
Adjust the pooled sample to 1 M NaCl. This will effectively strip electrostatically
bound ampholytes from proteins by ion exchange. Then dialyze into the buffer
appropriate for further uses.
3. Ammonium sulfate precipitation of proteins may also be effective in removing
ampholytes from samples.
4. Any number of chromatographic techniques, such as gel filtration, ion
exchange, hydroxylapatite, affinity chromatography, or use of AG 501-X8 resin,
can be used to separate proteins from ampholytes.
Section 9
Troubleshooting Guide
This guide is designed to answer common Rotofor cell questions. For further
information, please contact your local Bio-Rad representative. In the U.S., our
Technical Service department is available Monday to Friday, from 7:00 am to 5:00 P.M.
Pacific Time to answer all of your technical inquiries involving Bio-Rad equipment and
reagents. You can reach us by dialing 1-(800)-4BIORAD.
9.1 Solubility and Precipitation of Proteins
1. By definition, a protein at its isoelectric point (pI) has no net charge. Because little
charge repulsion exists between focused molecules, hydrophobic interactions
between proteins become predominant causing proteins to aggregate.
Maintaining the solubility of proteins in this case requires overcoming protein-protein
interactions. Several agents promote protein solubility. Detergents provide a
hydrophobic environment for proteins to mask interprotein interactions. Disulfide
bridges also may form between proteins leading to aggregation. This effect may
be overcome by the addition of reducing agents to the focusing media. Because
the solubility of proteins varies greatly, there is no one answer to the problem of
insolubility. Generally, the easiest method of getting proteins to remain in solution
is to add nonionic detergents, zwitterionic detergents, and/or chaotrophic agents
to the sample mixture.
In addition, glycerol from 5-25% (v/v) in the sample is highly effective for
maintaining the solubility and stability of proteins. Glycerol stabilizes water
structure and the hydration shell around proteins.
Table 9.1. Recommended Solubilizing Agents for the Rotofor System
Non-Ionic Zwitterionic Reducing Chaotropic
Detergents Detergents Agents Agents
0.1-3.0% Digitonin 0.1-3.0% CHAPS DTE 5-20 mM 1.0-8.0 M Urea
0.1-3.0% Octylglucoside 0.1-3.0% CHAPSO DTT 5-20 mM 0.1-2.0% Glycine
0.1-3.0% Triton X-114 BME 1-5 mM 0.1-2.0% Proline
2. When the solubility of a protein depends on maintaining high ionic strength during
focusing, increasing the concentration of Bio-Lyte ampholytes up to 5–8% in
your sample will help keep proteins in solution.
20