Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter - Manual (Page 15)

Native PAGE
Preparative Native PAGE gels are usually eluted with non-denaturing buffer systems (see
Section 7.4 for buffers useful in non-denaturing elution). The buffer initially used in running
the preparative Native PAGE gel should be the first non-denaturing buffer tried for elution.
In contrast to SDS-PAGE where proteins migrate according to size only, the mobilities
of proteins in native PAGE systems depends on both their charges and sizes. There is no sin-
gle elution/electrophoresis buffer system that will optimally elute all native proteins. When
selecting conditions for the elution of native proteins it is important to consider the pI of the
protein under investigation relative to the pH of the buffer system.
The most important consideration for optimum elution/electrophoresis of a protein is the
pH of the buffer. The pH of the buffer must be within the pH range over which the proteins
under study are stable and retain biological activity. In addition, the pH of the chosen buffer
system must impart sufficient charge to the protein for it to move through the gel at a rea-
sonable rate during the run.
The pH of the elution buffer determines the charges (and shapes) of proteins. Therefore,
during native elution/electrophoresis, the pH of the buffer will affect the migration of the pro-
teins. For example, a buffer with an alkaline pH value relative to the pI of a particular pro-
tein will impart net negative charge to the protein. In such a buffer system, the protein migrates
toward the positive electrode (anode). Elution buffers with acidic pH values relative to the pI
of a protein impart net positive charge to the protein so that it migrates toward the negative
electrode (cathode). A buffer with a pH value identical to the pI of a protein results in net
neutral charge on the protein and it will not move at all in an electric field. Buffers with pH
values far from the pI of the proteins result in fast elution/electrophoresis rates.
4.2 Running Conditions
The McLellan buffers described in this manual are a good starting point when selecting
an elution buffer (Section 7.4). 2 Eluting with any one of these buffers will give similar volt-
age and current readings based on their similar ionic strengths.
The elution rate of any given protein will vary depending on its pI and the pH of the elu-
tion buffer. As a general guide the buffer should be at least two pH units away from the pI of
the protein to impart sufficient charge to the protein. This must be tempered when the stabil-
ity and biological activity of the protein must be maintained by a given pH range. Proteins
bound with SDS will have a net negative charge. Under these conditions elution is best in a
high pH buffer. Determining the proper elution time will require following the optimization
procedure detailed below (Section 4.3).
Elution Constant Elution
Buffers Current Time
Large Eluter 200–250 mA 10–30 min
Small Eluter 75–100 mA 10–30 min
Note: Elution parameters are based on use of the McLellan buffers in the pH range of
8.1–10.2 to elute E. Coli lysate from a 1 mm thick 12% SDS-PAGE gel. A total of 200
µg of protein was loaded per gel. Elution times will vary with buffer pH, protein pI, gel
thickness, and gel porosity.
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