Bio-Rad PROTEAN II XL Cell - Manual (Page 28)

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Section 10
Two-Dimensional Electrophoresis
Two-dimensional electrophoresis can provide exceptionally high resolution of the protein components in a
complex sample. It is capable of resolving several thousand individual protein species. Based on the method
of O’Farrell (O'Farrell 1975), the first dimension is isoelectric focusing (IEF), during which proteins are
separated according to their isoelectric points. The second dimension is SDS-polyacrylamide gel
electrophoresis, in which proteins are separated on the basis of their molecular size. Since O’Farrell’s original
work, many variations of the 2-D procedure have been reported which may also be used. The following
procedure is based on the work of Dr. Denis Hochstrasser (Hochstrasser et al. 1986, Hochstrasser et al.
1988). The flow chart (Section 10.1) outlines in sequence the essential steps of 2-D electrophoresis and
refers to the solution protocols in Section 14.
Note: This section focuses on 2-D electrophoresis. The PROTEAN II xi cell may also be used for one-
dimensional tube gel electrophoresis. One can adapt this protocol to any of the common electrophoretic
techniques, using either continuous or discontinuous buffer systems, by following the instructions for
casting, loading, and running tube gels.
10.1 Sequence of Steps for 2-D Protocol
Step Time Interval
Day 1
1. Pour tube gels polymerize 2 hours
2. Prepare electrolytes, prepare and load samples
1⁄2 hour-1 hour
3. Electrophorese at 200 V constant voltage 2 hours
4. Electrophorese at 500 V constant voltage 2 hours
5. Electrophorese at 800 V constant voltage 16 hours (overnight)
6. Cast slab gels for second dimension gels while
first dimension gels are running 1 hour
7. Prepare second dimension running buffer 10 minutes
Day 2
8. Disassemble tube apparatus
2 minutes
9. Extrude gels from tubes and overlay tube gels
onto slab gels 25 minutes
10. Electrophorese the second-dimension SDS gel 4-41⁄2 hours
10.2 Protocol for IEF First Dimension
Casting IEF Tube Gels
For reproducible 2-D gels, it is essential that the IEF tube gels be precisely the same length and that
polymerization be identical from day to day. Care must be taken in pouring the gels to the same height so that
the polymerization height will be the same from tube to tube. An overlay step is not necessary in IEF first
dimension tube gels. The meniscus formed on top of the gel will not influence the pH gradient or the resolution
of the bands. The advantage of not overlaying is the formation of gels of more uniform length and composition.
Stock solutions and formulations for first dimension tube gels are given in Section 14.