Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 21)

5. An example of the importance of using the proper ampholytes in a fractionation is
demonstrated by a purification of Japanese water moccasin snake venom. The
protein of interest has a pI of 6.1 as determined by IEF gels. Bio-Lyte ampholytes
pH 6-8 were used for the initial fractionation. The fractions were analyzed and the
ones containing the specific protein were pooled. After refractionation, the fractions
were again analyzed on IEF slab gels and multiple bands were observed in all
fractions. When the same snake venom sample was initially fractionated in 5-7 Bio-Lyte
ampholytes the results were dramatically different. After refractionation, the protein
of interest was almost completely free of contaminating proteins. The conditions for
both experiments were identical except for the initial Bio-Lyte range; however,
much greater purity was obtained from the experiment using the 5-7 Bio-Lyte
ampholytes.
7.2 Sample Capacity
Choosing between the Standard Rotofor Chamber and the Mini Rotofor
Chamber is a matter of sample size. The Standard Rotofor Chamber is designed to
optimally fractionate milligrams to grams of total protein. The Mini Rotofor chamber is
designed to fractionate micrograms to milligrams of total protein. The smaller volume
of the Mini Rotofor decreases sample volume and is best suited for use with samples
of low protein concentration. Protein concentrations should be adjusted for desired
yield or to provide convenient assays of focused material, assuming each component
will focus in 1–3 channels, approximately equal to 3 ml/fraction in the Rotofor and
800 µl/fraction in the Mini Rotofor.
For example, if Rotofor fractions are analyzed by SDS-PAGE on a Mini-PROTEAN ®
II cell (catalog number 165-2940) and silver stained, the sample should contain a
minimum of 50.0 µg per component. More sensitive assays, such as activity assays,
decrease the necessary protein load.
The maximum protein load varies with the solubility of each sample and must be
determined empirically. However, preparative fractionation of 51 ml of lyophilized
cell culture supernatant containing 2.4 g of protein has been successfully performed
using the Rotofor Cell.
7.3 Power Conditions
We recommend running the Rotofor cell at constant power. During the initial
fractionation the voltage values will vary between samples depending on the relative
concentration of proteins and salts. The Mini Rotofor should be run using 12W constant
power and standard Rotofor should be run using 15W constant power.
When voltage is applied to a system of ampholytes and proteins, all the
components migrate to their respective pIs. In electrofocusing, the higher the voltage
the better the resolution. The limiting factor in achieving high resolution is how
efficiently electrically generated heat can be dissipated.
In a constant power mode, voltage gradually increases as the components focus.
The progress of the run is easily monitored by observing the voltage increase over
time. When the sample is focused, voltage levels off at a maximum. Runs typically
last 2-4 hours at 12 watts constant power and can require up to 3000 volts.
7.4 Cooling
Sample temperature affects activity and resolution. Many proteins, especially
enzymes, are temperature labile. The water recirculation chiller should be set about
10 °C cooler than the temperature required to maintain stability of your protein. The
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