Environmental Engineering Reference
In-Depth Information
of single cells enabled analysis of single cell type proteins [
36
]. In 1982, the use of
immobilised pH gradients (IPG) was introduced [
37
]. This method was then
improved and adapted for 2-DE. It became commercially established with special
convenient equipment and is the most commonly used 2-DE technique today. IPGs
are available as strips and for different pH ranges.
15.1.5 Advancement in Two-Dimensional Gel
Electrophoresis
In 1995, an improved version of original 2-DE technique was used [
38
]. The main
feature of this method is the use of large (40-30 cm) but thin capillary (1-D:
0.9 mm, 2-D: 0.75 mm) gels. They used carrier ampholytes for pH gradients
since IPGs in long distance gels formed protein bands rather than spots due to the
fixed pH gradient. The problem was resolved by running IPGs in a series of strips
with narrow pH gradients covering the whole gradient from pH 2-10 [
39
]. However,
the resulting 2-DE patterns had overlapping border lines in neighbouring pH ranges
making it difficult to recognise and eliminate the overlapping spots. In further
development to the technique, protein sample was applied on the acidic side of
the IEF gel and the run was stopped before the basic proteins moved out of the gel.
In this way the very acid as well as the very basic proteins were retained in the 2-DE
pattern, thereby saving the second 2-DE run for very basic proteins according to
O
s NEPHGE method [
40
]. Using this large-gel 2-DE method, more than
10,000 protein spots could be detected in one gel depending on the kind of tissue or
cells investigated, on the method of sample preparation and the protein detection
method.
The development of software for detection and statistical analysis of 2-DE spots
was another important expansion of the 2-DE technology. With the development of
the 2-DE technology, the great gap between protein detection and further analysis
of the proteins, i.e. large-scale protein identification, became obvious. This gap was
overcome at the beginning of the 1990s by developments in mass spectroscopy
(MS) allowing the identification of protein spots of 2-DE gels [
41
]. For 2-DE
experts, the 2-DE protein pattern was the endpoint and MS was just a tool to
identify all the protein spots resolved while for MS-experts, MS was the endpoint
and 2-DE just a tool for prefractionation of complex protein samples. MS-experts
pointed that 2-DE does not detect low-abundant proteins and hydrophobic mem-
brane proteins, and that 2-DE cannot be standardised up to the level, where
reproducibility is given from one laboratory to another.
Detection of low-abundant proteins by 2-DE can now be achieved by strategies
like prefractionation of cell and tissue proteins, large-gel 2-DE and fluorescent
labelling techniques for protein detection. For separating membrane proteins spe-
cial 2-DE methods are available [
42
]. However, translating 2-DE techniques into an
automated, high-throughput, MS adapted pipeline has not been feasible yet (Tecan
Farrell
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