Environmental Engineering Reference
In-Depth Information
method improvement led to the second step also being performed electrophoreti-
cally [
5
-
7
]. Introduction of polyacrylamide as matrix—the PAGE [
8
] improved the
electrophoretic separation of proteins considerably. Raymond, in 1964, was the first
who used PAGE for 2-DE separation of proteins [
9
]. The differences between the
first and the second separation steps were the pH, the composition of the running
buffer and the gel concentration.
Margolis and Kenrick [
10
] modified 2-D PAGE in order to separate the proteins
first according to charge and then according to molecular weight (MW). A very low
acrylamide concentration (2.7 %) was used in the first step, while an acrylamide
gradient (4-24 %) in the second step. In 1970, Kaltschmidt and Wittmann [
11
]
developed a 2-DE technique for separation of basic proteins that used Tris/EDTA-
Na
2
/borate-buffer, pH 9.6 and 8 % polyacrylamide in the first dimension and
KOH/acetate buffer, pH 4.6 and 18 % polyacrylamide were used in the second.
Extracts from 70S ribosomes of Escherichia coli were separated into more than
50 spots.
Disc electrophoresis was introduced in 1-DE technique by Ornstein [
12
] and
Davis [
13
]. This technique composed of a gel system in which the proteins migrated
throughout a stacking gel before entering the separation gel. The protein bands
became surprisingly sharp, and consequently much more proteins could be
detected.
Further two new methods that increased the resolution of proteins drastically
were introduction of IEF in polyacrylamide gel and SDS-PAGE. IEF not only
increased protein resolution but also the separation of proteins according to only
one parameter, the isoelectric point (pI). In 1970, the disc electrophoresis system
was used in the presence of SDS. SDS treatment gave proteins a negative charge
and, therefore, separation in an SDS gel depended only on the molecular weight of
the proteins. SDS-PAGE also improved the resolution of proteins considerably,
similar to IEF. The two one dimensional techniques, IEF- and SDS-gel electropho-
resis offered optimum conditions for the development of a 2-DE technique that led
to high resolution in both directions, and separation according to different criteria,
pI and MW. The development of the modern 2-DE began with the combination of
IEF in the first and SDS PAGE in the second dimension, a technique independently
developed by Dale and Latner, in 1969 [
14
] and Macko and Stegemann [
15
]. The
analysis of the mixture of serum with this method, Dale and Latner detected about
60 protein spots. Domschke et al. [
16
] used a technique similar to that of Macko and
Stegemann and separated serum proteins into about 90 spots. In the middle of the
1970s, several 2-DE techniques were published, all based on the concept to
combine IEF, SDS-PAGE and disc electrophoresis. Anderson and Anderson [
17
]
developed their own version and detected about 300 protein spots in 2-DE patterns
of human serum (plasma) proteins.
After 1975, the application of 2-DE expanded considerably. The possibility for
clinical applications made this method attractive. Other applications of 2-DE
include protein separation from particular cellular fractions, such as erythrocyte
membrane proteins [
18
], ribosomal proteins, chromosomal nonhistone proteins [
19
]
and, moreover, proteins of economic interest such as potato proteins [
20
] or wheat
