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Silver or Blue: What’s the Best Stain for Your Proteins?

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.

Silver stain

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.

Classical 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

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.

Bibliography

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.

4 Comments

  1. user-73738 on February 11, 2013 at 2:59 pm

    Yes, what Min has said about microwaving your gel is right. It helps you save a lot of time but you have to be careful so that you don’t overheat the gel or it might dehydrate, other than that it has no detrimental effect on the gel.

    And also you can do chloroform staining prior to coomasie staining if you have tryptophan residues in your protein, as chloroform binds to tryptophan and fluoresces under UV light. So it can be used as initial screening of your protein bands. You just have to soak the gel in chloroform for 1 min wash it MQ water until the chloroform is removed and then view it under UV light.

  2. Min on January 17, 2013 at 4:49 am

    Great article Laura. I just wanted to pitch-in a little bit more information here and there. First, regarding the colloidal Coomassie staining, there is a method dubbed “blue silver” by Giovanni Candiano published in the journal of Electrophoresis (2004) 25 1327-1333 that has a sensitivity in the 1 ng range and is less time consuming than you alluded to. With this method, I get perfectly stained gels in just 4 hrs (3 x 10 min for washing gels in MQ water, 3 h staining with the “blue silver”, 30 min destaining with MQ water). Second, the classical Coomassie staining can be accelerated by microwaving the gel until the stain or destain solution just begins to boil and let the gel rock at RT for 5 min, each time. Just a few tips.

    • Laura Fulford on February 3, 2013 at 4:16 pm

      Thanks for that extra info Min! I didn’t know you could microwave your gel, does it ever have a detrimental effect on the gel or any downstream applications?

  3. Firas on September 29, 2012 at 8:34 pm

    Thank you for the very interesting post. You didn’t touch on “Ponceau S” staining which I really like for checking complete transfer of WB, and to a lesser extent to detect equal loading of protein samples.

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