Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 28)

2. Clarify all sample solutions before focusing. Membrane cores are composed of
polyester membranes with a pore diameter of approximately 10 µm. The membrane
core can become clogged with insolubles in sample solutions. Starting solutions can
be clarified by centrifugation. To clean the membrane core, soak it in detergent,
dilute NaOH, or sonicate. Rinse the core well in distilled water after cleaning. For
complete removal of residual base, assemble the Rotofor cell with the membrane
core and prerun the cell with H
2
O until the voltage stabilizes. Then discard the water
and add sample. Generally the membrane cores will last at least 20 runs if they are
well cared for.
3. Protein samples that contain a charged detergent, like SDS, may experience a
shift in apparent pI and migrate to one or another end of the cell as a result of
acquired net charge. Use only non-ionic or zwitterionic detergents for this reason.
Some proteins are inherently associated with phospholipids, heme groups, or
other charged groups that affect the electrophoretic migration of proteins in a
pH gradient. A means must often be found to neutralize the effects that these
charged groups have on proteins during focusing. For example, the non-ionic
detergent, digitonin, has been found to be effective at disassociating negatively
charged phospholipid from integral membrane proteins. Digitonin provides a
suitable hydrophobic environment for maintaining the stability and biological
activity of these proteins during focusing in the Rotofor cell.
9.5 Power Conditions
Voltage is the driving force behind isoelectric focusing. Maximizing the voltage is
the best way to increase resolution. The cooling finger is capable of dissipating up
to 15 W of power generated in the large focusing chamber. The main factor limiting
voltage is efficiency of heat dissipation. These are common problems related to the
application of power.
1. Voltage fluctuations are caused by air bubbles trapped between the sample
and the ion-exchange membranes. Remove the assembled Rotofor core, hold
it vertically and tap on it to dislodge the bubbles. Turn the cell 180° and repeat
the bubble removal process. The minimum running volume of sample solution
should not be less than 35 ml for the standard focusing chamber and 18 ml for
the mini chamber.
2. Voltage decreasing at the beginning of the run is normal. At the beginning
of a run mobile charge carriers migrate through the chamber creating a relative-
ly high initial current. Eventually, desalting ceases and the pH gradient forms. As
the run proceeds, the resistance of the focusing medium increases and voltage
climbs. Do not set a limit on the voltage below 2,000 volts. When the voltage
finally plateaus, steady state has been achieved. Let the run continue for an
additional 15-30 minutes, then harvest.
3. Arcing between the anode and/or the cathode contact plate(s) and the contact
assembly(s) may occur for either of two reasons: 1) the solid brass points of the
contact assembly(s) are worn down and electricity is jumping across the gap, or
2) there is a leak in the coolant from the cooling finger housing and the coolant is
making the electrical connection. Either replace the contact assemblies left side
(catalog number 100-3780) or right side (100-3790) or repair the leaking cooling
finger with new O-rings from the Cooling Finger Repair Kit (catalog number
170-2954).
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