User Manual

C.B.S. Scientific Electro-Eluter Concentrator
9
5. Buffer Cycling System Set-up. This unit is designed to maximize yield by preventing any pH
gradient from forming inside the block by constantly re-cycling buffer. Attach tubing to outlet
on side of the third chamber to dual peristaltic pump. Split outlets from pump with “Y”
connectors, followed by attachment to the tubing connectors mounted in the safety covers.
The flow of recycled buffer drops out of anode and cathode buffer chambers into the mixing
chamber, and is then returned via the pump system. The remixed buffer drops onto the small
plastic strips, which protect the platinum wire. This prevents unwanted small air bubbles from
re-entering the buffer.
Buffer Re-Circulation
6. Running conditions for
a) Proteins prepared for Sequencing: Electrophoresis is carried out as per Hunkapillar
et. al.
b) Elution of Protein from SDS-Acrylamide Gels, not to be Sequenced: Buffer: 50mM
Tris Acetate (pH 7.8), 1mM EDTA, Current: 60-70 Volts, Duration: 15-20 hours
7. At the end of electrophoresis remove the blocks carefully from the chamber. Pipette off and
discard most of the running buffer in the loading and cross channels. Re-suspend the eluted
sample on the node membrane, typically 100-200ul of buffer (500ul in the 5ml block). For
initial runs save all of the buffer in the collection side above the conical chamber. If the
elution is incomplete, protein sample may not have migrated all the way down the membrane.
Therefore, check yield before discarding collection side buffer. See diagram on page 10.