Bio-Rad Mini-PROTEAN® Tetra Cell - Manual (Page 21)

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to their molecular weights. Proteins are separated according to
their molecular weights, making this system extremely useful for
calculating molecular weights.
3.3 Native PAGE
Native PAGE is a technique for separating biologically active
proteins. In contrast to SDS-PAGE, the mobilities of proteins in a
native PAGE system depend on both size and charge. There is
no single electrophoresis buffer system that will optimally purify all
native proteins. Key parameters for separating proteins in a native
PAGE system are isoelectric point (pI) of the protein of interest and
the pH of the electrophoresis buffer.
pH and pI
The pH of the electrophoresis buffer must be within the pH range
over which the protein of interest is stable and retains biological
activity. In addition, the pH of the buffer must impart sufficient
charge to the protein for it to move through the gel. Changes in
pH will affect both the charge and size (hydrodynamic volume) of
the protein of interest and will affect migration rates. For example,
a buffer with a pH greater than the pI of the protein will impart
a negative charge on the protein and it will migrate toward the
positive electrode (anode). Conversely, a buffer with a pH lower
than the pI of the protein will impart a positive charge and the
protein will migrate to the negative electrode (cathode). A pH equal
to the pI will result in no net charge in the protein and it will not
migrate in an electric field.
Protein mobilities are best modified by the buffer’s pH. Buffers with
a pH closer to the pI will provide the best resolution. However run
times may be lengthy. Conversely, buffers with a pH further from
the pI will migrate quickly but resolution may be compromised. The
choice of pH becomes a tradeoff between separation and speed.
How to Choose a Native PAGE System
1. Discontinuous Buffer Systems (Ornstein and Davis 1964)
This discontinuous buffer system should be the first
nondenaturing gel system tried. Detailed protocols are provided
in section 4.2. The advantage of a discontinuous system is the
use of a stacking gel to concentrate dilute protein samples.
However, the stacking phenomena can also cause aggregation
of some proteins and interfere with resolution. If protein
aggregation occurs, a continuous buffer system should be used.