Bio-Rad Mini-PROTEAN® Tetra Cell - Manual (Page 19)

15
Section 3
Separation Theory and Optimization
3.1 Introduction
Polyacrylamide gel electrophoresis separates molecules in complex
mixtures according to size and charge. During electrophoresis
there is an intricate interaction of samples, gel matrix buffers, and
electric current resulting in separate bands of individual molecules.
Hence the variables that must be considered in electrophoresis
are gel pore size, gel buffer systems, and the properties of the
molecule of interest.
Gel Pore Size
Gel pores are created by the crosslinking of polyacrylamide with
bis-acrylamide (bis) to create a network of pores. This structure
allows the molecular sieving of molecules through the gel
matrix. Gel pore size is a function of the acrylamide monomer
concentration used (%T). By convention, polyacrylamide gels are
characterized by %T, which is the weight percentage of the total
monomer including the crosslinker. The %T gives an indication of
the relative pore size of the gel. In general, pore size decreases
with increasing %T.
%T is calculated using the following equation.
%T = g acrylamide + g crosslinker x 100%
total volume (ml)
%C is the ratio of the crosslinker to the acrylamide monomer ratio
in the monomer solution. %C is calculated using the following
equation.
%C = g crosslinker x 100%
g acrylamide + g crosslinker
2.67% C is traditionally used for most analytical gels.
Gels can be made as a single continuous percentage throughout
the gel, or can be cast as a gradient %T through the gel. Typical
compositions are from 7.5% up to 20% for single percentage gels,
or gradients ranging from 4–15% to 10–20%.