DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 11

radioactive tag added, or they can be identified by immunoblotting (also
called western blotting).
Identification of proteins in gels by staining
A number of reagents can be used to nonspecifically or selectively
stain proteins, thus allowing the visualization of protein bands or spots
in gels.
Coomassie brilliant blue
Coomassie blue is a common dye used for protein chemistry be-
cause of its ability to bind to proteins, as well as other substances. Pro-
teins are stained in the PAGE gel following treatment of the gel with a
methanol/acetic acid solution, which causes the proteins in the individ-
ual bands to precipitate. Coomassie blue staining is not highly sensitive:
the detection limit is about 0.3 to protein in a band. However, for
many screening purposes, Coomassie blue dyes are adequate to reveal
the complexity and abundance of proteins in gels.
Ponceau S (Ponceau red)
Ponceau red differs from Coomassie blue staining in that it can be
used to stain proteins after transfer to a membrane, and provides a
rapid way to “visually” quantitate proteins on the membrane. Staining
with Ponceau red is reversible, and the proteins can then be character-
ized by Western blotting (see below) following destaining of the mem-
brane. Ponceau red is 1 to 2 orders of magnitude more sensitive than
Coomassie blue.
Silver staining
Silver can be used to stain proteins because of the ability of silver to
bind to chemical sidechains in the amino acids, including carboxyl and
sulfhydryl groups. Silver staining has a much greater sensitivity than
Coomassie or Ponceau staining, and can detect as little as 2 ng pro-
tein. It is commonly used to stain proteins separated by 2-dimensional
electrophoresis since the small amount of material recovered in the sec-
ond dimension, especially when IEF and SDS-PAGE are combined, can
often not be detected by other, less sensitive methods. Because silver
staining is irreversible, it sometimes creates problems when these spots
are sequenced or otherwise characterized by mass spectroscopy.