Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Methods for Staining Proteins in Gels

Staining of Proteins separated in electrophoretic gels with silver complexes was first used in 1972 [197], but the full potential of this method was realized later [359]. Protein silver-staining Methods are 100–200 times more sensitive than those based on Coomassie Brilliant Blue. Among the six Procedures proposed for this reagent, the best ones achieved a detection limit of 0.5 ng protein/mm² of PAG surface [282]. The sensitivity of silver staining varies somewhat depending on the properties and Nature of the protein, as demonstrated in a comparative study of four different protocols applied to human parotid saliva proteins; the choice of the optimal Staining Procedure largely determines the accuracy of data interpretation [115]. Electropherograms stained with Coomassie and silver complexes often differ from each other, and certain proteins, such as calmodulin and troponin C, are detected by Coomassie but fail to stain with silver complexes. It was found that such proteins require preliminary glutaraldehyde fixation for silver staining, and the corresponding washing procedure for the electropherograms must be strictly controlled to prevent the loss of soluble proteins [328]. Basic proteins are less sensitive to silver staining than neutral ones, and dual-staining methods combining Coomassie and silver salts have been proposed for them [176].

Silver complexes have successfully enabled the detection of Peptides in SDS-PAGE obtained from nanogram quantities of protein [222]. The study concluded that this method is faster, cheaper, and safer than radioactive labeling coupled with autoradiography or fluorography. Commercial reagent kits from Upjohn Diagnostics were used for 1.5 mm thick gels [324], and a protocol for staining thinner gels using a Bio-Rad kit as described in [408] was recommended.

The Mechanism of interaction between silver complexes and proteins involves phosphate [326], sulfhydryl, and carboxyl groups of Amino Acids [286]. Protein detection sensitivity with this reagent was enhanced following preliminary Treatment with glutaraldehyde [281] or formaldehyde [408], as well as prior reduction with dithiothreitol [269]. A study by [99] concluded that the sensitivity is enhanced exclusively by glutaraldehyde treatment (rather than formaldehyde) and that staining intensity correlates linearly with the number of Lysine residues in the protein.

The original version of the method [252, 359] has been modified by many researchers to simplify it and reduce costs [175, 253—255, 269, 281, 305, 324, 408]. See the critical review [305].

Silver staining is the first reliable method for detecting Histones in Triton-acid-urea gels, exhibiting a nearly linear relationship between staining intensity and protein amount within the 0—50 ug range [259].

It was shown that sequentially immersing PAG electropherograms in hyper- and hypotonic solutions makes it possible to detect as little as 10 fg of protein using silver complexes [285]. At the same time, the original silver-staining procedure is multistep, labor-intensive, and involves numerous manual operations. Incomplete removal of unreacted silver complexes can lead to high Background coloration. An extended washing procedure for electropherograms in a flowing liquid helps avoid these complications and yields a linear response in the range of 2–70 ng of sample per gel band, as demonstrated for Muscle protein [126]. A novel microwave drying technique for PAG has been proposed [124], which reduces the process time to 3.5 min (instead of the usual 35–45 min) for 0.75 mm thick gels and to 1 h for 3 mm gels (compared to 4–5 h required by the Bio-Rad gel dryer).