Technical Support FAQ

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Technical Support FAQ


Question:

I'm getting uneven harvesting. What should I do?


Answer:

There are a number of reasons why you can get uneven harvesting with the Rotofor and Mini Rotofor focusing chambers:

Note that the two end compartments of the focusing chamber are slightly larger than the other chambers, and therefore hold more liquid in their fractions. It is expected that the two end fractions will be larger than the others.

We specify a vacuum that pulls at 5 to 10 mm of mercury. If the vacuum is not this strong or stronger, the harvesting may be uneven. Check the needles in the harvesting block and the harvest block lid. They should not be plugged, or unequal in length. Check the plastic harvest tubing. It should not be kinked or plugged. If necessary, soak the tubes in Bio-Rad cleaning solution, Triton X-100, or 0.01 M NaOH to remove residual matter; then rinse the tubes with ddI water. CAUTION: DO NOT insert all of the needles into the water at the same time with the vacuum on, as the harvest lid may crack! Dry the tubes, one at a time, by aspirating the fluid from within them with a vacuum hose.

There is a best order of the tasks to obtain good harvesting of fractions:

  1. When the run is over, put the toggle switch to harvest.
  2. Set up the harvesting rack with 20 tubes, one in each hole in the rack inside the vacuum chamber.
  3. Make sure the hose is attached to the vacuum source and to the vacuum port on the vacuum chamber of the harvesting apparatus.
  4. Remove both covers from the IEF chamber. The top port must be removed to obtain even harvesting.
  5. Turn on the vacuum and set the lid onto the vacuum chamber. Make sure each needle is positioned over each tube.
  6. Position the needles under the IEF chamber directly under the tape covering the lower port.
  7. With your thumbs on the top of the chamber, and your fingers holding the needle array in place just below the tape, press the needle array up quickly and firmly through the tape into the chamber. The fractions will be evacuated within about 1 or 2 seconds. Note that it may be useful to practice the harvesting procedure several times before doing a run to get the procedure down.

The compartments of the focusing chamber may contain unequal volumes at the end of the run if you do not fill the focusing chamber full. As proteins become focused, the osmotic pressure in each Rotofor system 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 sample chamber completely filled.


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