Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 417

The intrinsic complex nature of peptides makes it mandatory to choose a highly
selective and specific technique that determines potency, stability, and purity of a
compound. Alhough remarkable progress has been achieved in the field of protein
analysis, much concentrated effort is required to further enhance the selectivity and
specificity of existing analytical methods.
8.7.3.4 Silver Staining
Along with popular dyestuffs like coomassie blue, R and G types, and several fluo-
rescent dyes, one of the very popular techniques of protein staining and identification
is silver staining. Silver staining has been widely documented in the literature as a
method of choice for protein staining following gel electrophoresis. This technique was
introduced back in 1979 by Switzer, Merril, and Shifrin [42] . The method is highly
sensitive and can fairly detect proteins in concentration ranges as low as 0.3-10 ng.
The underlying principle of silver staining is based on a donor acceptor mecha-
nism involved in complexation as well as redox mechanisms. Silver ions, when added
to a protein sample in the form of silver-based dye, combines with electro donor or
lewis bases of amino and sulfydryl groups present in amino acid fragments of the
proteins. The silver undergoes reduction in the presence of these electron donors
and is converted to free metallic silver, which has a characteristic color and can be
visualized as spots wherever the reduction takes place [43] . Silver staining proto-
cols comprise the following two methods predominantly: (1) silver amine or alkaline
methods and (2) silver nitrate or acidic methods [44] . The choice of method usu-
ally depends upon the limit of detection, particularly the signal-to-noise ratio. Silver
amine or alkaline method has the advantage of lower noise levels or, in other words,
higher sensitivity; however, it is tedious and cumbersome. The second method, based
on acidic reagents, is relatively faster and less time consuming but less sensitive as
well. With technological advances over a period of time, a large number of methods
have been published in the literature that offer advantages over these existing classi-
cal methods in terms of precision, time duration, compatibility, and accuracy [45,46] .
To augment the precision and accuracy of silver staining methods, they have been
used in combination with mass spectrometry and similar techniques. One of the
key steps to improving the efficacy of the silver staining technique is fixation using
formaldehyde or glutaraldehyde. These chemicals act as cross-linking agents and are
responsible for cross-linking of two lysine residues within proteins. The fixation is a
critical step prior to mass spectrometric analysis. Formaldehyde/glutaraldehyde fixa-
tion largely eliminates the trouble of trypsin digestion and reduces subsequent pro-
tein extraction from the gel [47] .
Materials and instrumentation involve the use of ultra pure water for prepara-
tion of all reagents, buffers, and wash solutions. The solutions required to be used in
protocol are discussed as below:
1. Fixation solution: This usually composed of 50% ethanol, 12% acetic acid, and 0.05%
formalin. The chemicals are dissolved and final volume is adjusted using deionized or