Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 17)

4.3 Fraction Collection
1. Load the test tube rack with twenty 12 x 75 mm culture tubes and place it inside
the harvest box. Place the lid on the box, making certain that each stainless steel
collecting tube is inside a test tube. Connect a vacuum source to the vacuum port
on the box and turn on the vacuum to hold the lid in place. A vacuum pump or
house vacuum of 10–50 mm Hg is recommended.
2. When focusing is completed, move the black toggle switch to the HARVEST
position. This stops the cell rotation with the cell properly aligned for sample
collection, i.e., with the alignment pins and taped collection ports on the bottom
of the focusing chamber. All manipulations which follow the end of rotation
should proceed as quickly as possible to minimize mixing.
3. Turn the power supply off, disconnect the power supply, remove the cover, and
move the Rotofor cell and the harvesting box next to one another. Remove both
the upper and lower focusing chamber cell cover blocks. Mount the needle array
on the two alignment pins on the bottom of the chamber. Grasp the needle array
with the fingers of both hands while placing the thumbs on the top of the focusing
chamber. Take care not to block any of the uppermost ports. Quickly push the
needles firmly and uniformly all the way through the sealing tape into the chamber.
This will cause all 20 fractions to be simultaneously aspirated from the cell and
delivered to the collection tubes.
Fig. 4.2. Harvesting samples after focusing is complete. Make sure thumbs do not cover the
uppermost ports.
4. Turn off the vacuum source and remove the test tube rack. Note that all the
odd numbered fractions are in one row and the even numbered fractions are in
the other row of the rack.
4.4 Refractionation
The fractions containing the protein of interest may also contain other, contaminating
proteins after the initial fractionation. Refractionation of Rotofor fractions is one way to
increase sample purification. Because of its lower volume requirement the Mini Rotofor
chamber is ideal for refractionating pooled fractions.
13