Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 38)

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Section 9
A Guide to Preparative Native-PAGE
9.1 Introduction
Conventional gel elec trophoresis buffer sys tems and media are used with the Model 491 prep cell to separate
in di vidual components from their nearest con tami nants. This guide describes a method for selec ting the best
nondenaturing PAGE system to isolate a particu lar protein with the Model 491 prep cell.
Native-PAGE Theory
Preparative native-PAGE is a technique for high yield purification of bio logi cally active molecules. In contrast to
SDS-PAGE where proteins migrate ac cording to size only, the mobili ties of proteins in native-PAGE systems
de pend on both their charges and sizes. There is no single electrophoresis buffer sys tem that will opti mally
purify all native proteins. When selecting conditions for the purification of a native pro tein there are some basic
pa rameters to con sider: the pI of the pro tein under investigation, and the pH of the electrophore sis buffer
system.
In preparative native-PAGE, the most important consideration for op timum reso lution of a protein is the pH of
the electrophoresis buffer. The pH of the elec trophoresis buffer sys tem must be within the pH range over which
the protein under study is stable and re tains its biological ac tivity. In addition, the pH of the chosen buffer system
must impart sufficient charge to the pro tein for it to move through the gel at a reasonable rate during the run.
Changes in pH alter the charges (and shapes) of proteins and there fore will af fect the migration rates and
resolution patterns of proteins in the sample. For exam ple, a buffer with an alkaline pH value relative to the pI
of a particu lar protein will impart net negative charge to the pro tein. In such a buffer sys tem, the protein migrates
towards the positive electrode (anode). Electrophoresis buffers with acidic pH values relative to the pI of a
protein impart net positive charge to the protein so that it migrates towards the neg a tive electrode (cathode).
A buffer with a pH value identical to the pI of a pro tein results in net neutral charge on the protein and it will not
move at all in an electric field.
In native-PAGE, protein mobilities are best modified by the buffers pH. Electrophoresis buffers with pH values
close to the pI of the protein of interest will theoreti cally provide the best resolution. However, the resultant
migration rate may be too slow for elution from the prepara tive gel column. Conversely, buffers with pH values
farther away from the pI of the protein of interest re sult in faster mi gration rates, but, with a loss of resolution.
The choice of pH be comes a com promise between separation and speed.
Once the native protein is purified, an enzyme assay or immunoblot can be used to identify the specific location
of the protein in a slab-gel or in the fractions collected from the Model 491 Prep Cell. Analysis by SDS-PAGE
can be used to confirm the resolution and purity. Silver stained SDS-PAGE gels will demonstrate the presence
of any contami nating pro teins.
9.2 How to Choose Native-PAGE Systems
Discontinuous Buffer Systems
The discontinuous buffer system of Ornstein-Davis (Tris/chloride/glycine) should be the first nonde naturing gel
system tried (Ornstein 1964). Detailed protocols are pro vided in Section 9.3 for using this system. In discontinuous
systems, the buffer used in the stacking gel, resolving gel and in the electrode chambers differs in ionic
concentration and pH. An ad vantage of discontinuous systems for dilute protein so lutions is the use of stacking
gels to concentrate the sample. However, the stacking phenomena encountered in discontinuous systems can
cause aggrega tion of some pro teins and this can severely in terfere with reso lu tion. Section 9.2 presents an
alternative to using discontinuous buffer systems.
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