Environmental Engineering Reference
In-Depth Information
bands can be further analysed by mass spectroscopy analysis and results can be
predicted from database accession number. Typically 200-500 protein spots can be
observed and identified in a silver stained 2D gel. 2D PAGE provides more detailed
analysis of protein samples compared to 1D SDS PAGE and offers advantage of
loading higher protein amount compared to the 1-DE gel. Main limitations are
inability to identify hydrophobic membrane proteins. Mixtures of proteins are
separated by two properties in two dimensions on 2D gels, i.e. charge and size.
2-D electrophoresis begins with 1-DE but then separates the molecules by a second
property in a direction perpendicular from the first. In 1-DE, proteins (or other
molecules) are separated in one dimension, so that all the proteins/molecules will
align along a lane but that the molecules are spread out across a 2-D gel. Because it
is unlikely that two molecules will be similar in two distinct properties, molecules
are more effectively separated in 2-DE than in 1-DE. The two dimensions used to
separate the proteins are isoelectric point and protein complex mass in the native
state.
To separate the proteins by isoelectric point is called isoelectric focusing (IEF).
Thereby, a gradient of pH is applied to a gel and an electric potential is applied
across the gel, making one end more positive than the other. At all pH values other
than their isoelectric point, proteins remain charged. If they are positively charged,
they are pulled towards the more negative end of the gel and if they are negatively
charged they are pulled to the more positive end of the gel. The proteins applied in
the first dimension move along the gel and accumulates at their isoelectric point;
that is, the point at which the overall charge on the protein is 0 (a neutral charge).
For the analysis of the functioning of proteins in a cell, the knowledge of their
cooperation is essential. Most often proteins act together in complexes to be fully
functional. The analysis of this sub-organelle organisation of the cell requires
techniques conserving the native state of the protein complexes. In native poly-
acrylamide gel electrophoresis (native PAGE), proteins remain in their native state
and are separated in the electric field following their mass and the mass of their
complexes, respectively. To obtain a separation by size and not by net charge, as in
IEF, an additional charge is transferred to the proteins by the use of Coomassie
Brilliant Blue or lithium dodecyl sulphate. After completion of the first dimension
the complexes are destroyed by applying the denaturing SDS-PAGE in the second
dimension, where the proteins of which the complexes are composed of are
separated by their mass. As a separation, detection, and quantitation technique,
2-DE is an important tool, especially for clinical laboratories involved in the
determination of protein expression levels and disease biomarker discovery
(Fig. 15.2 ).
Before separating the proteins by mass, they are treated with sodium dodecyl
sulphate (SDS) along with other reagents ( SDS-PAGE in 1-DE). This denatures the
proteins (that is it unfolds them into long, straight molecules) and binds a number of
SDS molecules roughly proportional to the protein
s length (when
unfolded) is roughly proportional to its mass, and thus attaches a number of SDS
molecules roughly proportional to the mass of protein. Since the SDS molecules are
negatively charged,
s length. Protein
'
'
the result of this is that all of the proteins will have
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