Biomedical Engineering Reference
In-Depth Information
4.4 SDS-PAGE
Electrophoresis is a well-known method for the separation and analysis of complex
protein mixtures where charged molecules dispersed in a fluid migrate under
electric field. Capillary electrophoresis (CE) and gel electrophoreses (1D or 2D)
are two widely employed methods used for analyzing NP-protein complexes.
SDS-PAGE and Western blotting are qualitative methods which are based on
comparison, and therefore, getting quantitative data out of them is not easy. In
general SDS-PAGE can detect between 1 and 50 ng of a single protein band [
8
].
4.4.1 Capillary Electrophoresis
Separation of proteins by CE can be selected using a UV or fluorescence detector
[
16
]. This method was used to study the adsorption of albumin onto poly(methoxypo-
lyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) nanoparticles. CE allowed
direct quantification of adsorbed proteins without the requirement for a desorption
procedure. However, the detection sensitivity is not very high.
4.4.2 One-Dimensional Gel Electrophoresis
Proteins migrate through a vertical gel of acrylamide and bisacrylamide and are
separated according to their size due to their different electrophoretic mobility
(Fig.
4.3
). The proteins required to be denatured and to be negatively charged,
simply by boiling them with a reducing agents (DTT or beta mercaptoethanol) and
anionic detergents. This treatment of the proteins by SDS causes protein repulsion
and thus detachment from the NP surface. The proteins resolved in the gel can be
stained (Coomassie brilliant blue, silver nitrate staining, deep purple, etc.),
and densitometry analysis is routinely used to quantify protein abundance [
17
].
SDS-PAGE (polyacrylamide gel electrophoresis) is an extremely quick and cheap
technique. It suffers from poor protein separation if the protein complex is too rich,
resulting in co-migration in the same gel bands of several proteins.
4.4.3 Two-Dimensional Gel Electrophoresis
Two-Dimensional Gel Electrophoresis (2D-GE) is a powerful technique in proteo-
mics where more than 5,000 proteins can be separated within the same gel
[
16
]. Proteins are separated in two dimensions. In the first dimension the proteins
are separated according to their charge by isoelectric focusing (IEF) and in the
