Environmental Engineering Reference
In-Depth Information
Studying cellular and tissue proteins from a global standpoint opened the door to
many interesting questions: How do protein composition and expression change
during embryonic development, cell differentiation and in the course of ageing?
How do the proteins from different organs, tissues and cell types differ? Is it
possible to study genetic variation between various individuals or different inbred
mouse strains on a broad spectrum of proteins as revealed by 2-DE? And what is the
effect on proteins if an organism is exposed to different environments or treat-
ments? These questions are now being answered with the advances and technology
developments in the 2-DE. The 2-DE technique was initially designed for the
investigation of the chemically induced point mutation effects on a broad spectrum
of tissue proteins. Mutations of single nucleotides frequently led to amino acid
substitutions, which induced charge alterations in the corresponding protein. The
mutated proteins can then be detected in 2-DE protein patterns by alterations in
their electrophoretic position. To avoid any restrictions in the electrophoretic
mobility of the native proteins carrying charge alterations due to mutations, SDS
was not used for the second dimension. Under these electrophoretic conditions
275 protein spots were detected in 2-DE patterns from embryonic mouse liver
[
22
]. Proteins were revealed by amido-black staining. In a modified version using
SDS in the second dimension, 440 spots were detected in protein patterns from
embryonic mouse liver [
23
]. Here Coomassie Blue staining was used for protein
detection. Analysis of 2-DE protein patterns in the course of development of mouse
embryos and studies on the effect of chemically induced mutations on proteins [
24
]
were among the first investigations of the 2-DE technique. The 2-DE technique was
established for separation of total proteins from
E. coli
. Cell culturing allowed
labelling of proteins with radioactive amino acids and protein detection by autora-
diography [
25
]. Using this method, O
Farrell obtained 2-DE patterns for 1,100
proteins which after staining with Coomassie Blue, however, decreased to only
about 400 spots [
25
].
2-DE was used to resolve complex protein extracts from HeLa cells [
26
], human
brain tissue [
27
] lymphocytes [
28
], trisomic cells [
29
], mouse embryos [
30
] and
other cells and tissues. By the early 1980s it was confidently stated that it would be
possible to detect 2,000-3,000 proteins with the present 2-DE technology [
31
]. For
the evaluation of large numbers of protein spots, computer programs were devel-
oped for the first time [
32
]. In later years, a plan to catalogue every protein produced
in the human body; “The Human Protein Index” was developed [
33
]. A systematic
analysis of the mouse protein complement, identification of the relevant genes was
performed and these probes were used to isolate the corresponding human
genes [
34
].
The sensitivity of detecting separated proteins in 2-DE gels was drastically
increased by the introduction of silver staining technique [
35
]. Later, fluorescence
staining with CyDyes enhanced the sensitivity of protein detection further and
allowed running test and control sample in the same gel. This improved the
reproducibility and quantification of protein patterns considerably. Saturation label-
ling, a special, highly sensitive technique of the CyDye system, could be used to
detect much less abundant proteins. Saturation labelling along with microdissection
'
