Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 18)

After screening the samples collected from the first fractionation, pool the fractions
containing the protein of interest. These pooled samples (typically 3–5 fractions) can
be reapplied either to the Rotofor or Mini Rotofor for refractionation. Rotofor fractions
obtained from an initial run contain ampholytes whose range spans the pI of the protein
of interest. It is best to add no additional ampholytes to the sample to be refractionated.
Because ampholytes and salts are not added prior to the refractionation, higher
voltages can be obtained because of the low ionic strength of the sample. High
voltages lead to better resolution during focusing. Upon refractionation, the
ampholyte range is much narrower and more specific to the protein of interest. The
pooled fractions contain a small part of the initial pH range which spans the pI of
the protein of interest. This is spread across the length of the chamber during
refractionation, providing a shallow pH gradient, and thereby increasing the likelihood
of obtaining one or more fractions of pure protein.
4.5 Final Purification
The Rotofor is designed to quickly separate proteins of interest from other proteins
in a sample. Bio-Rad’s Model 491 Prep Cell can purify individual proteins from Rotofor
fractions by continuous-elution electrophoresis. Conventional gel electrophoresis
buffer systems and media are used with the Model 491 Prep Cell. Using SDS-PAGE
or native-PAGE, the Prep Cell can isolate specific components from complex mixtures
containing micrograms to 200 milligrams of total sample. Up to 5 milligrams per band
can be resolved. Using SDS-PAGE the cell isolates molecules that differ in molecular
weight by 2%. Using non-denaturing PAGE the cell can isolate molecules that differ in
charge by 0.1 pH units. Electrophoretic purification can also be effective in removing
ampholytes from samples. See Section 12.
Section 5
Disassembly and Cleaning
1. Rinse the needle array and its associated tubing with water as soon as possible
after use. Do not use the vacuum box to pull water through the needle array.
This may damage the box. Rinse the box with water.
2. Take the focusing chamber from the stand. Loosen the nylon screws and
remove the cathode chamber.
3. Leave the cathode and anode chambers intact. The ion exchange membranes
must be stored wet. Remove the electrolyte and fill the electrode chambers with
distilled water. If properly stored, the membranes will not decrease in performance
between runs. Before starting a new run, the electrolytes must be replaced with
fresh solutions.
4. Loosen the nylon screws on the anode chamber and remove the focusing chamber
and membrane core. Rinse all chamber components with water and air dry. Do not
expose the focusing chamber to concentrated acid, base, or alcohol. The membrane
core requires additional care, especially if there has been protein precipitation during the
run. A spatula can be used to loosen and remove caked precipitates. Soak the membrane
screens in saline and then in detergent or 0.1 M NaOH to remove traces of protein. An
ultrasonic cleaner will facilitate the cleaning process. Finally, rinse the screens with
water. For complete removal or residual NaOH or other cleaning compounds, assemble
the Rotofor cell with the membrane core, add distilled water, and apply 5W power until
the current stabilizes. Then discard the solution and add the sample. Cleaning with
strong oxidizing agents, such as hypochlorite, or organic solvents should be avoided, as
they will damage the membrane core. If properly cleaned, the membrane core can be
immediately reused.
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