Staining proteins following SDS-PAGE or 2-dimensional electrophoresis is a very useful technique for visualising a global population of proteins or checking expression of your recombinant proteins, but how do you know which stain to use? This article explains the pros and cons of two of the most common protein staining techniques, silver staining and coomassie brilliant blue staining.
Need a high sensitivity stain with no requirement for downstream applications? Silver staining may just be what you need.
Silver staining utilises the protein binding properties of silver ions, which are then reduced to silver metal using a developing solution, creating a visible image. The primary benefit of silver staining is its high sensitivity, as it is able to detect less than 1 ng of protein (Weiss et al. 2009), making it extremely useful for applications involving low protein levels. However, silver staining involves multiple steps and reagents, making the process relatively time consuming and laborious. In addition, the gel requires developing after staining, in order to visualise the proteins, and the length of time required for developing is highly variable between gels, meaning reproducibility is low.
Another disadvantage of silver staining is that the use of formaldehyde when fixing the gel makes silver staining incompatible with mass spectrometry. Silver staining protocols do exist that are compatible with mass spectrometry, but at the sacrifice of sensitivity. Silver staining has a narrow linear dynamic range (the range at which the level of staining is linear to the concentration), making it less suitable for quantification.
Silver staining is my least favourite method of protein staining, not just because of the multiple steps required but also due to of the use of several dangerous chemicals (including silver nitrate and formaldehyde), which I like to avoid if possible. It is also really difficult (from my experience) to remove silver stains from benches and equipment (so beware!).
If you want more information on different silver staining techniques, including those for use with mass spectrometry, as well as protocols, see Chevallet et al. (2006) .
Coomassie brilliant blue (CBB)
Coomassie staining is probably the most well known protein staining technique. Two main types of coomassie staining exist, the original or “classical” coomassie and the more recently developed colloidal coomassie.
The classical coomassie staining technique involves incubating protein gels with a coomassie staining solution, which stains the whole gel, and not just the proteins. Destaining of the gel allows visualisation of protein bands, as the dye is retained better by the proteins than the gel. This technique is cheap and simple to perform, but is considerably less sensitive than the previously described silver staining, having a detection limit of only about 100 ng (Weiss et al. 2009), making visualisation of low abundance proteins difficult. The low reproducibility of classical coomassie staining is also a disadvantage of this method and is due to the difficulty in standardising the destaining step. One big advantage of coomassie staining is that it is compatible with mass spectrometry.
This method of staining is my personal favourite for simple tasks such as visualisation of recombinant proteins, or generated antibodies, as it so simple and quick to perform.
Colloidal coomassie provides a happy medium between classical coomassie staining and silver staining, having relatively high sensitivity and being simple to perform, as well as being compatible with mass spectrometry.
This method is an adaptation of classical coomassie staining using a modified coomassie dye (G-250 instead of R-250). This method of staining takes longer than the classical staining technique, but is also simple to perform. Colloidal coomassie has several advantages over traditional coomassie staining, including increased sensitivity, with a detection limit of around 10 ng (Weiss et al. 2009). As the colloidal dye does not penetrate the gel, no destaining is required (although destaining can be performed to improve background), resulting in higher reproducibility than silver staining or classical coomassie staining. Many different protocols for colloidal coomassie exist, and several of these are described and compared in Dyballa & Metzger (2012).
The increased sensitivity of this method over classical coomassie makes it ideal for experiments involving low protein levels, such as analysing binding proteins in co-IPs, especially when you’re considering identifying the protein bands using mass spectrometry.
A quick summary of the different stains is given in the table below.
Chevallet et al. 2006Chevallet, M., Luche, S. & Rabilloud, T. (2006), Silver staining of proteins in polyacrylamide gels, Nature protocols 1, 1852–1858.
Dyballa & Metzger 2012Dyballa, N. & Metzger, S. (2012), Fast and sensitive coomassie staining in quantitative proteomics, in K. Marcus, ed., ‘Quantitative Methods in Proteomics’, Vol. 893 of Methods in Molecular Biology, Humana Press, pp. 47–59.
Weiss et al. 2009Weiss, W., Weiland, F. & Görg, A. (2009), Protein detection and quantitation technologies for gel-based proteome analysis, in J. Reinders & A. Sickmann, eds, ‘Proteomics’, Vol. 564 of Methods in Molecular Biology, Humana Press, pp. 59–82.