Environmental Engineering Reference
In-Depth Information
hydrophobic proteins such as membrane-bound proteins, and also smaller proteins
and peptides (
15 kDa). The DIGE technique is also relatively expensive com-
pared to silver or Coomassie staining of gels. Although 2D-PAGE has been limited
by its inability to resolve proteins that are too basic or too acidic, too large or too
small, this limitation is continuously being worked at. For example, the separation
of basic proteins can be analysed using IPGs in the pH range of 4-12. The selection
of type of stain used to visualise protein depends on the conditions and goals of
experiment and the downstream applications.
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15.2 Parameters affecting 2D PAGE efficiency
Several parameters of sample or nanoparticles affects the analysis of sample using
2D PAGE technique. In the following sections these parameters are discussed in
detail one by one.
15.2.1 Particle Size and Porosity
The sizes and shapes of particle or nanoparticles are important factors that deter-
mine their physical and chemical properties. Capillary electrophoresis (CE) has
proved to be one of the most powerful separation techniques [
43
] and has been
applied to separate a variety of differently sized materials, including inorganic
oxide [
44
], latex [
45
], polystyrene, silicate [
46
] and gold particles [
47
]. The sepa-
ration mechanism relies upon surface charge of the material and hence their
separation behaviour is similar to that of charged molecular species in CE. The
variably sized particles display specific mobilities in a capillary column under the
presence of an external applied voltage because they have different charge-to-size
ratios. CE in an approach traditionally used to separate molecules in terms of their
sizes and shapes [
48
]. For studying the effect of size and shape of the nanoparticles,
silver nanoparticles were synthesised using microwave irradiation method [
49
]. An
aqueous suspension comprising a mixture of silver nanorods (~6.2 and 55.5 nm)
and nanoparticles (~80.2 nm) was obtained. Mafune and co-workers [
50
] and Chen
and Yeh [
51
] have demonstrated that the surfactant SDS has a positive stabilising
effect on silver nanoparticles.
During the CE separation process, samples prepared in a solution that differed
from that of the running electrolyte were immediately enveloped by the running
electrolyte. This blending offered the possibility of bringing particles into a defined
environment; any subsequent changes in the effective charge-to-size ratios of the
particles could be monitored by changing the composition of the running electro-
lyte. Concentrations of SDS surfactant in the range 0-40 mM were used to evaluate
the concentration dependence of the separation of a mixture of 17.0—and 49.7 nm
diameter silver nanoparticles in a single run. No separation occurred when a
