Bio-Rad Mini-PROTEAN® Tetra Cell - Manual (Page 20)

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The total monomer concentration for optimal separation is referred
to as optimal %T. Optimal %T will vary depending on the molecular
weight of the molecule of interest. Empirically the pore size
providing optimum resolution for proteins is that which results in a
relative mobility (Rf) value between 0.55–0.6. Rf values for specific
proteins are calculated as follows.
Rf = Distance migrated by the protein of interest
Distance migrated by the ion front
Gel Buffer System
The buffer system determines the power requirements and
affects separation. The buffer system is composed of the buffer
used in the gel and the running buffer. There are continuous and
discontinuous buffer systems.
Continuous Buffer Systems
In continuous buffer systems, the same buffer ions are present at
constant pH in the gel and electrode reservoirs. The gel is typically
made of one continuous %T and the sample is loaded directly into
the part of the gel where separation will occur. The band width is
determined in part by the height of the sample load, so samples
should be concentrated and volumes small for best results.
Discontinuous Buffer Systems
In discontinuous buffer systems different buffer ions are present
in the gel and electrode reservoirs. By using different buffers in
the gel and in the electrode solutions and adding a stacking gel
to the resolving gel, samples are compressed into a thin starting
band and individual proteins are finely resolved and separated.
Discontinuous buffer systems were devised initially for use with
undenatured, or native proteins; however the most popular
discontinuous system employed is the SDS-PAGE buffer system
by Laemmli (1970). Formulations for this system are included in
section 4.1.
3.2 SDS-PAGE (Laemmli) Buffer System
The Laemmli buffer system is a discontinuous buffer system
that incorporates SDS in the buffer. In this system, proteins
are denatured by heating them in buffer containing sodium
dodecyl sulfate (SDS) and a thiol reducing agent such as
2-mercaptoethanol. The resultant denatured polypeptides take on
a rod-like shape and a uniform charge-to-mass ratio proportional