Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 20)

Section 7
Optimizing Fractionation
7.1 Ampholyte Choice
1. Bio-Lyte ® ampholytes are complex mixtures of synthetic buffering electrolytes with
closely spaced pI’s and high conductivity. Bio-Lytes are supplied at concentrations
of 40% (w/v), except in the pH ranges 3-5 and 8-10, which are at 20%. The final
concentration of Bio-Lytes used in the Rotofor system depends on the protein
concentration in a given sample:
Protein per Bio-Lyte
milliliter Ampholytes
>2 mg 2%
1 mg 1.5%
0.5 mg 1%
0.25 mg 0.5%
2. Up to 8% (w/v) ampholyte concentrations have been used for various applications.
Ampholytes at 1% permit higher applied voltages and are recommended if
refractionation is not required. 2% ampholytes will provide greater buffering and
are necessary when refractionation is performed. If protein precipitation occurs
during the run because of the desalting effect of focusing, sample solubility may
be maintained with higher ampholyte concentrations. Use the following formula to
determine the appropriate volume (V
1
) of a 40% Bio-Lyte ampholyte solution to
give a desired final concentration in your Rotofor sample.
For the equation: (C
1
)(V
1
)=(C
2
)(V
2
), solve for V
1.
where: C
1
= Starting concentration of Bio-Lyte (40%)
V
1
= Unknown volume of 40% Bio-Lyte to give desired final
concentration
C
2
= Final or desired concentration of Bio-Lyte
V
2
= Final volume of the sample to be applied to the Rotofor
(35–58 ml) or Mini Rotofor (18 ml)
3. The pI of the protein of interest can be determined by running the sample on a
flatbed slab IEF cell (such as the Model 111 mini IEF cell) using the broad pH
range of 3-10. IEF markers (catalog number 161-0310) run in the same gel
allow the pI of the protein of interest to be estimated. Alternatively, the pI can
be estimated by running the sample in the Rotofor cell using a broad range
3–10 ampholyte. The pI of the protein of interest will correspond to pH of the
Rotofor fraction where the protein of interest focuses.
4. A narrow pH range of ampholytes spanning the pI of the protein of interest should
be used for the initial fractionation. Narrow range fractionation first separates the
protein of interest from the bulk of its contaminants. The pI of the protein of interest
should fall in the middle of the ampholyte range.
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