Biomedical Engineering Reference
In-Depth Information
mass. To check this proportionality a Ferguson plot should be
made (determination of the relative mobility R
f
of the protein of
interest in dependency of the total concentration of acrylamide%T)
or the exact mass is proved by ESI or MALDI mass spectrometry
2
.
The relative mobility of a macromolecule is given by the quo-
Relative mobility
tient of its distance of migration measured from the start of separa-
tion and the distance of electrophoresis front (position of tracking
dye):
distance
macromolecule
distance
tracking dye
=
R
f
If R
f
of different proteins should be compared, the calculations
have to be made from the same slab gel to eliminate variances
in acrylamide concentration, polymerization, and electrophoretic
conditions.
Whether a gel with polyacrylamide concentration gradient or
with homogeneous concentration, and whether a denaturating
(SDS and/or 2-mercaptoethanol and DTE/DTT
3
, or urea) or a non-
denaturating (native) system is used, depends on the analytical
objective. For a survey or for separation of a mixture of molecules
with a broad range of molecular weight, a gradient gel should be
chosen, especially because an increased protein sieving effect is
observed in the lower molar mass range leading to sharper bands,
whereas a homogenous gel should be applied if proteins similar
in size and charge will be analyzed. Concerning the geometry of
the gel, slab gels are preferred because it is possible to run several
samples in parallel under the same conditions.
To illustrate the resolution force of polyacrylamide gels a plot
of marker proteins separated in gels with different acrylamide con-
centrations is given in Fig. 2.1. This figure illustrates that gels
with homogenous acrylamide concentration separate over a broad
molecular range too, if the migration distance is long enough, but
it is observed that a band broadening occurs mostly at higher R
f
.
Thegelcompositionisoftendescribedbytheterms%Tand%C.
%T refers to the total content of acrylamide (sum of acrylamide and
cross-linking monomer), whereas %C is the part of cross-linking
substance (e.g., N,N
-methylene bisacrylamide) of monomers.
AA + C
V
=
%T
·
100
C
AA + C
·
=
%C
100
2
Discussions of different PAGE systems and possible errors are given by
Johnson. (Johnson G (1983) Gel Sieving electrophoresis: a description
of procedures and analysis of errors. In: Glick D (ed.) Methods in
biochemical analysis, vol 29. Wiley, New York, p 25).
3
DTE and DTT (Cleland's reagent) are stereoisomeres, which as well
as the optical pure substances or the racemate are effective reductants.
