Environmental Engineering Reference
In-Depth Information
Reproducible patterns were obtained at a ratio of 3:1 plasma to suspension [
70
]. The
particle concentration and related surface area for adsorption are important, and not
the suspension volume. It is recommended to admix a particle suspension as highly
as concentrated to plasma or serum to avoid too strong a dilution by the water of the
particle suspension, to avoid reducing artificially the solubility of some proteins by
the addition of water.
15.2.9 Method of Separation
The separation method is crucial to obtain reproducible results. Some of the
currently used methods are centrifugation [
71
], chromatography [
72
] and separa-
tion by magnetic fields [
73
]. The separation method can affect the pattern. Chro-
matography might lead to desorption effects at the end of the separation process.
The particle sediment from centrifugation needs to be washed to remove excess
plasma in the pellet. Normally, several washing steps need to be applied.
15.2.10 Quality of Water Used
Organic contaminants affect the polymerisation of acrylamide gels, resulting to
poor quality gels. The migration of proteins in the gel is dependent on ionic strength
and pH of the running buffer. The ionic strength and pH of buffers used to prepare
the gel and running buffer have to be maintained to assure reproducible results.
High levels of ions could alter the ionic strength of the solutions and thus affect the
migration rate of proteins. Water that is contaminated with bacteria and degradation
by-products such as proteases that may degrade proteins affect the ionic strength of
solutions leading to poor gel quality. Highly purified water is thus an essential
component to obtain rich gel quality, with lower background and better spot
resolution.
15.3 Applications of Gel Electrophoresis
in Pharmaceuticals
15.3.1 Protein Identification
The initial goal of most proteomics projects is to identify and determine differential
abundance of proteins or posttranslational modifications (PTMs) between samples
followed by detailed analysis of individual proteins of interest. 2-DE followed by
mass spectrometry provides direct visual confirmation of changes in protein/PTM
