Protein Protocols Protein Protocols

266 Dunn
particularly the ε-amino group of lysine (7), and with sulphur residues of cysteine and
methionine (8). However, Gersten and his colleagues have shown that “stainability”
cannot be attributed entirely to specific amino acids and have suggested that some
element of protein structure, higher than amino acid composition, is responsible for
differential silver staining (9).
Silver staining procedures can be grouped into two types of method depending on
the chemical state of the silver ion when used for impregnating the gel. The first group
is alkaline methods based on the use of an ammoniacal silver or diamine solution,
prepared by adding silver nitrate to a sodium–ammonium hydroxide mixture. Copper
can be included in these diamine procedures to give increased sensitivity, possibly by a
mechanism similar to that of the Biuret reaction. The silver ions complexed to proteins
within the gel are subsequently developed by reduction to metallic silver with formaldehyde
in an acidified environment, usually using citric acid. In the second group of
methods, silver nitrate in a weakly acidic (approx pH 6) solution is used for gel impregnation.
Development is subsequently achieved by the selective reduction of ionic silver
to metallic silver by formaldehyde made alkaline with either sodium carbonate or
NaOH. Any free silver nitrate must be washed out of the gel prior to development, as
precipitation of silver oxide will result in high background staining.
Silver stains are normally monochromatic, resulting in a dark brown image. However,
if the development time is extended, dense protein zones become saturated and
color effects can be produced. Some staining methods have been designed to enhance
these color effects, which were claimed to be related to the nature of the polypeptides
detected (10). However, it has now been established that the colors produced depend
on: (1) the size of the silver particles, (2) the distribution of silver particles within the
gel, and (3) the refractive index of the gel (11).
Rabilloud has compared several staining methods based on both the silver diamine
and silver nitrate types of procedure (12). The most rapid procedures were found to be
generally less sensitive than the more time-consuming methods. Methods using glutaraldehyde
pretreatment of the gel and silver diamine complex as the silvering agent
were found to be the most sensitive. However, it should be noted that the glutaraldehyde
and formaldehyde present in many silver staining procedures results in alkylation
of α- and ε-amino groups of proteins, thereby interfering with their subsequent chemical
characterization. To overcome this problem, silver staining protocols compatible
with mass spectrometry in which glutaraldehyde is omitted have been developed (13,
14) but these suffer from a decrease in sensitivity of staining and a tendency to a higher
background. This problem can be overcome using post-electrophoretic fluorescent
staining techniques (15). The best of these at present appears to be SYPRO Ruby (see
Chapter 35), which has a sensitivity approaching that of standard silver staining and is
fully compatible with protein characterization by mass spectrometry (16).
The method of silver staining we describe here is recommended for analytical
applications and is based on that of Hochstrasser et al (17,18), together with modifications
and technical advice that will enable an experimenter to optimize results. An
example of a one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) separation of the total proteins of human heart proteins stained
by this procedure is shown in Fig. 1. The power of 2-DE combined with sensitive