Environmental Engineering Reference
In-Depth Information
Fig. 15.2 Simplified 2D PAGE experimental setup. Image analysis is done by software and
matched with fingerprint available in protein database. Last two steps are additional to separate
and identify specific separated protein
approximately the same mass-to-charge ratio as each other. In addition, proteins
will not migrate when they have no charge (a result of the isoelectric focusing step)
therefore the coating of the protein in SDS (negatively charged) allows migration of
the proteins in the second dimension (SDS-PAGE, is not compatible for use in the
first dimension as it is charged and a nonionic or zwitter ionic detergent needs to be
used). In the second dimension, an electric potential is again applied, but at a
perpendicular angle from the first field. The proteins get attracted to the more
positive side of the gel (because SDS is negatively charged) proportionally to
their mass-to-charge ratio. This ratio remains nearly the same for all proteins.
The proteins progress is be slowed by frictional forces. The gel therefore acts like
a molecular sieve when the current is applied, separating proteins on the basis of
their molecular weight with larger proteins being retained higher in the gel and
smaller proteins being able to pass through the sieve and reach lower regions of
the gel.
The result of this is a gel with proteins spread out on its surface. These proteins
can then be detected by silver or Coomassie Brilliant Blue staining. Further
identification of the proteins can be done using in-gel digestion of protein spots,
and subsequent identification of the proteins by mass spectrometry.
15.1.4
2D PAGE History
The 2-Dimensional Electrophoresis (2-DE) based protein separation dates back to
the late 1950s. The first 2-DE technique was developed by Smithies and Poulik in
1956 [
3
]. They used filter paper for separating proteins in the first dimension while
in the second dimension proteins were separated on starch gel. Almost 20 human
serum proteins were resolved. Around the same time, another technique was
developed by Grabar and Williams [
4
]; they first performed agarose gel electro-
phoresis and in second step diffusion was performed against an anti-serum. Further
