Gel‐Staining Techniques – Dyeing to Know It All

Abstract

Staining techniques are the primary method for quantitative detection of gel‐resolved proteins. With stain sensitivity defining the extent of total protein detected, staining‐technique enhancements that facilitate increased detection of low‐abundance proteins are constantly sought. Although stains must exhibit high detection sensitivity, they must also be broadly applicable, practical, cost‐effective and compatible with downstream identification techniques. Currently, the most widely utilised in‐gel protein stains are colloidal Coomassie Brilliant Blue and SYPRO Ruby (or slight variants on each). As with any stain, each requires extensive characterisation and rigorous optimisation of protocols to effectively define limits of sensitivity and quantitative capacity. However, the procedures involved in preparing, resolving, imaging and analysing samples can also have significant consequences on quantitative outcomes, and thus must be fully considered in order to facilitate detailed and reproducible analyses (i.e. as required in top‐down proteomics).

Keywords: protein staining; quantitative analysis; gel electrophoresis; fluorescence; imaging; detection; proteomics; protein sample preparation

Figure 1. LDR comparison: SR and cCBB. Dilution series of a mixture of pure protein standards (seven in total) were resolved by 1DE, and stained with cCBB or SR. Bars represent the LDR, ‘*’ indicate an LDR wider than that of SR and ‘^’ indicates gel detection sensitivity beyond that of SR.Modified from Gauci et al. (2013) © Elsevier.
Figure 2. Total spot detection of mammalian and plant proteomes: cCCB and SR. (a) Total spot number analysis of mouse brain (solid bars) and A. thaliana plant leaf (hatched bars) proteomes. Error bars represent SEM (n = 3). Statistically significant differences in comparison to SR are denoted, where *p < 0.05, ***p < 0.0001 (Student's t‐test). (b) Distribution of all detected protein spots between SR and cCCB; i – mouse brain membrane protein, ii – mouse brain soluble protein, iii – A. thaliana membrane protein, iv – A. thaliana soluble protein. For each fraction and stain, total detected spots are bold in parentheses, with stain‐specific spots detected given immediately below and as a percentage of the largest number of spots detected (i.e. by cCCB in all conditions); intersections indicate numbers of overlapping protein spots detected by both stains.Modified from Gauci et al. (2013) © Elsevier.
Figure 3. 3DE of excised gel regions following 2DE of (a–c) whole liver and (d–e) whole cardiac muscle protein extracts from mouse. (a) Miniformat 2DE of whole mouse liver protein extract using 3–10 IPG. (b) Orthogonal resolution of ‘stacked’ proteins at the alkaline extreme (pH ∼10, highlighted in a) using large format (18 cm) gradient SDS‐PAGE. (c) Tris‐tricine SDS‐PAGE of oligopeptides migrating at the dye front (highlighted in a). (d) Miniformat 2DE of whole mouse cardiac muscle protein extract using 3–10 IPG. (e) Orthogonal resolution of excised high‐abundance regions (highlighted in d) to resolve obscured low‐abundance proteins. All gels were stained with SR. Modified from Butt and Coorssen (2005) © American Chemical Society.
Figure 4. 2DE‐resolved SR‐stained (a, b) soluble and (c, d) membrane proteomes from rat brain, (a, c) before and (b, d) after Deep Imaging. Reproduced from Wright et al. (2014) @ PLoS One.
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Further Reading

Gauci V, Wright E and Coorssen J (2011) Quantitative proteomics: assessing the spectrum of in‐gel protein detection methods. Journal of Chemical Biology 4: 3–29.

Merril CR (2005) Gel Staining Techniques (eLS). Chichester: John Wiley & Sons, Ltd, http://www.els.net (doi: 10.1038/npg.els.0003918).

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Wright EP, Partridge MA, Padula MP, et al. (2014) Top‐down proteomics: enhancing 2D gel electrophoresis from tissue processing to high‐sensitivity protein detection. Proteomics 14: 872–889.

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Mansour née Gauci, Victoria J, Noaman, Nour, and Coorssen, Jens R(Jun 2016) Gel‐Staining Techniques – Dyeing to Know It All. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002686.pub3]