Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 25)

3. Decreasing the protein load will also help keep the protein in solution. The
largest amount of protein concentration in solution that has been successfully
fractionated in the Rotofor cell is 4 grams. The lower limit for protein loading
depends on the sensitivity of your detection system.
9.2 Factors Affecting the pH Gradient
Non-linear pH gradients are rarely observed when sample is prepared properly
and the Rotofor cell and its parts are carefully maintained. A non-linear pH gradient
may be caused by one or more of the following:
1. Electrolyte leakage. Excessive leakage of electrolyte across the ion exchange
membranes into the focusing chamber will decrease the number of fractions on
the linear portion of the pH gradient and reduce the effective voltage across the
sample. To determine if this is occurring, check the pH of the fractions.
Alternatively, fill the Rotofor focusing chamber with distilled, deionized water
and run the Rotofor at 12 W constant power. If the amperage does not
decrease to < 6 mA and the voltage does not increase to near 2,000 V within
5–10 minutes, the chances are good that you have electrolyte leaking into your
sample. Some common causes are:
A) Expired Vent Buttons. Vent buttons lose their capacity to vent the gases
produced during electrolysis over time and when there is too much electrolyte
in the chamber. The pressure that results within the electrolyte chambers
forces electrolytes into the focusing chamber. Replace the vent buttons (catalog
number 170-2957) every 4 to 5 runs.
B) Worn O-rings and/or electrode Gaskets. The Rotofor repair kit contains
replacement parts for these items (catalog number 170-2953). Lubricating
the O-rings with a small amount of silicone O-ring grease or Cello-Seal
™
will extend their useful lifetime (catalog number 170-2954).
C) Cracked, dehydrated, or worn out Ion-exchange Membranes (catalog number
170-2956). These last 4 to 5 runs.
2. Uneven harvesting. Variations in the volumes of harvested fractions may affect
the linearity of the collected pH gradient. Be sure to remove both harvesting and
loading port covers before piercing the sealing tape with the harvesting block
needles. Also make sure that the harvesting tubes are clean and clear of blockages
by soaking in Bio-Rad cleaning concentrate (catalog number 161-0722) or dilute
0.05 M NaOH and rinsing well with DDI H
2
O after each run. Dry the tubes by
aspirating each individual tube with a vacuum line. Be careful not to block the
loading port holes with your fingers during harvesting.
The compartments of the focusing chamber contain unequal volumes at the
end of the run. As proteins become focused the osmotic pressure in each
Rotofor compartment may vary. If the focusing chamber is not completely full,
this may cause unequal distribution of fluids in the 20 compartments. This
effect will vary as a function of protein load and concentration of solubilizing
additives. Reproducibility of results, especially where isolation of a protein in a
particular fraction number is expected, will depend on the constancy of these
factors. To alleviate the osmotic effect, the Rotofor cell should be run with the
focusing chamber completely filled.
3. Premature harvest. Too short a run will result in a partially-formed pH gradient
and poorly focused proteins. The Rotofor cell is normally run for 3 to 6 hours.
To assure complete focusing, continue the run for 1/2 hour after the voltage
stabilizes, then stop the electrophoresis and harvest the focused protein.
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