Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 26)

4. High salt sample. The salt (or buffer) concentration in the sample may be too
high. This will decrease the effective voltage across the sample and may reduce
the number of fractions on the linear portion of the pH gradient. Resolution is
dependent on both high voltage and maximizing the number of fractions on the
linear gradient. If a particularly high ion concentration is necessary to preserve
the stability and/or activity of your protein, Bio-Lyte ampholytes (which are ionic
molecules) may be substituted for salts. For example, preparation of the enzyme
aldose reductase (pI ~ 5.0) from porcine lens for purification using the Rotofor
cell required that the protein sample be at low ionic strength (< 1.0 mM buffer)
to maximize voltage and resolution
10 . Since aldose reductase is unstable under
these conditions, the following procedure was used to avoid exposure to low
ionic strength:
1.0 ml of 5% Bio-Lyte ampholytes, previously fractionated using the Rotofor cell at
4.5 to 5.5 pH range, were added to 1.0 ml of 5.0 mg / ml protein solution in a 10.0
mM phosphate buffer. This solution was exhaustively dialyzed against 25.0 ml of
the same 5% Bio-Lyte solution, thereby making the final phosphate concentration
less than 1 mM. The salt concentration in the sample was reduced to a reasonable
level while the ionic strength required to maintain the stability of the enzyme was
retained.
If the pH gradient plateaus or dips near the middle, this may be due to the presence
of excess buffer in the protein sample solution. The pH of the gradient will be
buffered at the pK of this buffer, creating a dip or plateau in the gradient in this
region. The symptom may be many fractions with the same pH. Reduce buffer
salts to < 10 mM.
5. High sample temperature. At 12 W, the temperature inside the chamber is generally
10 degrees higher than the temperature of the circulating coolant. The cooler the
run, the more stable the proteins will be. 4 °C is the optimum sample temperature.
6. Non-reproducible pH gradients. Use sufficient concentration of ampholytes.
Batches and brands of ampholytes may also vary. Do not run the Rotofor cell
more than 1–2 hours after voltage stabilization. Reduce salt to below 10 mM.
Always run the sample at or below 4°C. Check the integrity of the Bio-Lyte
ampholytes. Ampholytes should be stored at 4°C in the dark. Guaranteed shelf
life of opened Bio-Lyte ampholytes is 1 year.
9.3 Recovery of Biological Activity
1. pH. Some proteins are especially sensitive to rapid pH shifts and to extremes of
pH that exist at the extreme ends of the Rotofor focusing chambers. To avoid
exposing your protein to these potentially damaging pH extremes during initial
focusing, “prefocus” the focusing media (i.e. Bio-Lyte ampholytes, additives,
water, etc.), without protein, for about an hour. This will establish the pH gradient.
Then inject your protein sample into the sample chamber at or near the point in
the pH gradient that corresponds either to the pH of the protein sample solution
or to the pI of your protein of interest. Addition of your protein sample solution in
as small a volume as possible decreases exposure to rapid pH shifts and pH
extremes, minimizes the amount of time the protein spends in the Rotofor cell
and maintains native tertiary structure.
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