Biomedical Engineering Reference
In-Depth Information
Fig. 2.1.
Semilogarithmic plot of molecular weight (M
r
) of marker proteins vs relative mobility
(R
f
) of marker proteins in gels of different acrylamide concentrations %T. Proteins: 1 aprotinin
(6.5 kD); 2 lysozyme (14.5 kD); 3 soybean trypsin inhibitor (21.5 kD); 4 carbonic acid anhydrase
(31 kD); 5 hen ovalbumin (45 kD); 6 bovine serum albumin (66 kD); 7 phosphorylase b (97.4 kD);
8
β
-galactosidase (116 kD); 9 myosin (205 kD)
AA: amount of acrylamide (g); C: amount of cross-linker (g); V:
volume of gel mixture (ml).
Self-made PAGE gels should have at least 50 mm separation dis-
tance and a width of 0.75 - 1 mm. Generally, for analytical purposes
slab gels of 60
1 mm are sufficient, but also larger gels are
usable, especially for semi-preparative purposes.
The electrophoresis is controlled by a power supply mostly in
the constant current (cc) or constant voltage (cv) mode. It is advised
against application of constant current during separation, because
duringtheruntheelectricresistanceincreases,andbecausecurrent
is constant, voltage and heat production increase, too.
Most of the electrophoresis systems are sensitive to ionic
×
80
×
Salt effects in PAGE
strength (“salt content”) of samples. Dialysis of the sample against
a 100-fold volume of sample buffer for 30 min or protein precipi-
tation by TCA and dissolving of the pellet in sample buffer obviate
this problem.
PAGE systems described in this chapter are well established;
nevertheless, modifications concerning acrylamide concentration
(%T as well as %C) may be done to optimize the separation condi-
tions.
Caution!
Acrylamide is very toxic. Avoid inhalation of dust and
wear gloves during manipulation with monomers!
