Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 19)

Section 6
Sample Preparation
6.1 Salt Concentration
1. Samples should be desalted (e.g., by dialysis or Bio-Gel ® P-6 chromatography)
prior to ampholyte addition to insure that the nominal pH range of the ampholyte
will extend over the full length of the focusing chamber and that the maximum
voltage can be applied. It is best to limit salt concentrations in the samples to
about 10 mM for optimum fractionation. However, the maximum salt capacity
will vary with the application, therefore optimum running conditions should be
determined empirically. During focusing, all salts migrate to the compartments
next to the anode and cathode, effectively desalting the sample.
2. The sample should not be in a buffer greater than 10 mM concentration.
Buffers add to the conductivity of a sample and decrease resolution. Also,
buffering solutions may flatten the pH gradient in the region of the pK
a
of the
buffer.
6.2 Clarification
Turbid sample solutions should be clarified by filtration or centrifugation to
remove extraneous cellular debris that might clog the membrane core.
6.3 Solubility
If the solubility of proteins presents a problem, adjusting the sample to 3–5 M
urea is recommended. Higher urea concentrations, up to 8M urea, can be used.
Be sure to deionize the urea using AG
® 501-X8 mixed bed ion exchange resin
(catalog number 143-7424). Addition of non-ionic detergents, such as: CHAPS,
CHAPSO, octylglucoside, digitonin, or Triton X-114 is also valuable in maintaining
the solubility of focused proteins. The concentration of detergents used is usually
between 0.1% and 1%. Alternatively, solubility can sometimes be maintained by
increasing the Bio-Lyte ampholyte concentration in the sample. Check the solubility
of the protein by diluting it in detergent or urea and running it on an analytical IEF
gel. If the protein does not show signs of precipitation in the IEF gel, it should not
precipitate in the Rotofor cell.
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