A Blue Dive: From 'Blue Fingers' to 'Blue Silver'

A Comparative Overview of Staining Methods for In-Gel Proteomics

Isabella Panfoli, Daniela Calzia, Laura Santucci, Silvia Ravera, Maurizio Bruschi, Giovanni Candiano

Expert Rev Proteomics. 2012;9(6):627-634. "Download"

Abstract and Introduction

Abstract

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of 'blue silver' colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and 'blue silver' CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the 'blue silver' method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented 'blue silver' can evidence the complexity of the sample.

 

Introduction

As was foreseen, in-gel proteome analysis has continued to progress, exploiting its immense potential.[1] In particular, since the pioneering work by O'Farrell,[2] 2D electrophoresis (2-DE), based on the physical parameters isoelectric point (Ip) and molecular weight (Mr),[3,4] has been established as a mature technique for large-scale proteomic analysis and has challenged gel-free multidimensional protein separation technologies.[5] This is especially true since the advent of mass spectrometry (MS) and of databases that have facilitated the characterization of proteins.[6] The expression of thousands of proteins can be detected in a single 2-DE gel. 2-DE technology is extremely informative for the study of functional expression and post-translational modifications of protein isoforms.[7] Despite some limitations, mostly with some particularly hydrophobic proteins,[8] development of immobilized pH gradients[9–12] has rendered the technique fast, and so reliable as to separate proteins differing by a fraction of isoelectric point (pI). By contrast, in gel-free proteomic methods, which analyze peptides, information about Mr and pI is lost and quantitative analysis depends on stable isotope labeling.[13] The improvements in technologies for gel detection and mass analyzers over the last decade urges us to consider to what extent staining methods address the appropriate characteristics of sensitivity, quantitative attributes (detection limit, linear dynamic range) and compatibility with MS analyses.[14,15] Detection is a prerequisite to the identification of spots. It can be achieved through staining of proteins in a sample. In general terms, a staining reagent consists of an organic or inorganic chemical dissolved in an appropriate solution. The reversible or irreversible binding of the dye to the proteins precipitates or changes color or emits fluorescence, allowing detection of the protein under visible or ultraviolet light.[16] Staining methods based on diversified principles have been optimized and can be essentially divided into visible or invisible, and pre- or post-electrophoresis stains.[14]


Visible Staining Procedures

Coomassie Brilliant Blue

Coomassie brilliant blue (CBB) stain remains the preferred dye for quantitation in electrophoresis[1,16–18] owing to its ease of use, full MS compatibility and linearity over approximately one order of magnitude. Coomassie owes its name to a town in Ghana, where British troops won a battle in 1873. Some of us still remember being able to recognize each other as members of the first electrophoresis society in the early 1970s, thanks to the blue color of the tips of our fingers due to the habit of pulling gels out of the tray without wearing gloves. The authors were named 'blue fingers' after this. Among Coomassie dyes there are the Coomassie blue R-250 and G-250, disulfonated triphenylmethane, used by the biologists. The detection limit is 10–100 ng.[19,20] CBB is still in use since its first application in 1963 to stain proteins separated by cellulose acetate electrophoresis.[21] It is believed to interact with proteins by Van der Waals hydrophobic noncovalent interactions and also through electrostatic bonds with the amino groups of proteins in acidic environment. In classical protocols, CBB is used up to 1% in the presence of an alcohol (typically methanol) and acetic acid. As the dye has a lower affinity for the gel matrix than for proteins, destaining is performed in a similar solution.[18] It has been reported that heating dramatically decreases the time required for CBB staining and destaining of SDS-PAGE gels. For example, at 70°C, staining of a 1.5 mm gel can be completed in 5 min while destaining can be completed in 20 min.[22] An environmentally friendly method to destain CBB has been developed, allowing the disposing of CBB in manner and recycling the destaining solution. Various types of adsorbent papers (Kimwipes, multifold towels and Whatman number 1 and 3 filter paper) adsorb CBB. Kimwipes have been reported to work best[14] and the method is useful in reducing organic liquid waste.


Colloidal CBB

Colloidal CBB staining came later on, thanks to the attempts to reduce the background staining. Diezel et al. observed that CBB-G in 12.5% Trichloroacetic acid is converted into a colloidal[23] hindering dye entry into the gels, therefore giving a low background. Neuhoff et al. was the first to develop a staining method using colloidal CBB-G in the presence of ammonium sulfate and phosphoric acid, demonstrating the benefits of adding alcohol to the staining solution.[24] This is allowed the detection of weak spots.[19,20] The addition of methanol in the classical staining solution (20% methanol, 1.6% phosphoric acid, 8% ammonium sulfate, 0.08% CBB G-250), intended to shift the equilibrium from the colloidal form to dispersed dye, and facilitated the staining;[24] however, this introduced a main critical point, as will be discussed below. The principal characteristics that make colloidal CBB an excellent staining method are its ease of use, extremely low signal-to-noise ratio and its sensitivity.[16] Colloidal particles are in equilibrium with dispersed dye in solution. Protein staining (and low background) is achieved when the free dye enters the gel, while the colloidal dye particles are excluded. Lately, Candiano et al.,[25] Wang et al. [26] and Pink et al. [27] proposed different protocols aiming to overcome the limit of detection of CBB staining, still requiring approximately 10 ng of protein in a band. 'Blue silver' was developed by Candiano et al. as a modification of Colloidal CBB increasing dye by 20% and phosphoric acid by fivefold (staining solution: 20% methanol; 10% phosphoric acid; 10% ammonium sulfate; 0.12% G-250).[25] The lower pH, causing protonation of Asp and Glu residues, is thought to promote more efficient hydrophobic binding to aromatic and hydrophobic residues, allowing a more intensive staining. Blue silver is more sensitive than the other colloidal CBB-G stains, with a detection limit approaching 1 ng for BSA. The possibility of destaining 'blue silver' exclusively with ultrapure water is also advantageous as it is low in cost and minimally affects stained bands. Another modified protocol was described by Wang et al.:[26] after fixing, staining solution employs 5% v/v acetic acid, 45% v/v ethanol and 0.125% (w/v) CBB R-250. Indeed, colloidal CBB-G staining is more sensitive than CBB-R in solvent solutions (8 ng).[14,18] The procedure requires multiple steps. Moreover, the need for two destaining solutions (first 5% v/v acetic acid, 40% v/v ethanol; then 3% v/v acetic acid, 30% v/v ethanol) renders the method prone to dye loss also by the stained proteins. Pink et al. [27] substituted acetic acid with phosphoric acid during the fixation (10%) and staining of the gels (8%) and also 5% aluminum sulfate (Al2(SO4)3) 14–18 hydrate in the staining solution. Al2(SO4)3 is used in the fabric industry as a mordant; however, Pink et al. stated that it also acts as an ion-bridging reagent, as Al3+ binds the dodecyl sulfate part of sodium dodecyl sulphate (SDS), by coagulation. Destaining is carried out in 2% phosphoric acid and 10% ethanol; afterwards gels are put in water overnight. Pink et al. modified this approach using a solution containing 10% ethanol, 0.02% of CBB G-250, and 8% phosphoric acid and 5% aluminum sulfate.[27]

Scanning CBB fluorescence with an infrared laser scanner was reported to provide an increase in sensitivity of two orders of magnitude, setting this method to a sensitivity as high as twice that of

SYPRO Ruby (SR), and therefore the best in the field.[1,15,28] A less irritating but sensitive CBB stain (detection limit about 12 ng) was developed (CGP). CGP employs citric acid, CBB G-250 and polyvinylpyrrolidone. Unlike CBB, it should avoid in vitro methylation of proteins.[29]


Silver Stain

Silver-staining protocols are the most sensitive, nonradioactive protein staining methods.[16] These are based upon deposition of metallic silver on proteins.[16,18] Typically they are multistep, time-consuming procedures that can, however, detect 100 pg to 1 ng protein per spot.[1,30] Alkaline and acidic silver nitrate stains can be distinguished. Numerous variants are available for both. Acidic silver nitrate staining was shown to have a detection limit of 2–4 ng.[16] Ammoniacal staining is more sensitive for basic proteins.[31] As it is not an end point procedure, silver staining shows poor linearity with saturation effects and a narrow dynamic range (one to two orders of magnitude), and thus is not reliable for quantification. Moreover, silver staining detects the proteins mainly on the gel surface[16] and the presence of glutaraldehyde in the classic silver staining method[31] causes modification of peptides, rendering the procedure incompatible to MS applications. Even though omission of glutaraldehyde in acidic silver-staining renders the procedure compatible to MS applications, in general silver stain is not compatible with MS, which is always true for ammoniacal silver stains.[16]


Curcumin Stain

A nontoxic fluorescent/colorimetric reversible extremely cheap protein stain has been developed that utilizes heat-solubilized Curcumin, the yellow pigment from Curcuma longa (turmeric). Interestingly, even though Curcumin is insoluble in water and ineffective in staining proteins if dissolved in ethanol, when heated at 100°C in water it is soluble up to 1.5%. Staining is achieved within 30 min. Authors report sensitivity similar to that of CBB. Destaining is not required and excess dye can be freely disposed of.[8]


Alta Staining

A simple, cheap and rapid protein staining method for polyacrylamide gels during electrophoresis (or for nitrocellulose during blotting) was reported. It utilizes a 5% solution of Alta, a commercially available cosmetic preparation, added in the anodic buffer during electrophoresis. After run, the gel is washed in ultrapure water and can be viewed under visible light. There is no need for destaining.[32]

 

Negative Stains

Negative, or reverse, stains yield transparent colorless protein bands on an opaque gel background.[16] These methods are the fastest to apply (they can take as little as 5–15 min); however, they appear neither accurate nor linear. Staining is mostly due to metal (zinc or copper) interaction with SDS in the areas where proteins are absent, with a sensitivity lying between that of CBB and silver. Metals precipitate to produce a negative image. For example, a negative staining with CuCl2 was developed: immediately after electrophoresis run, the gel is incubated in 0.3 M CuCl2 in the presence of 10–100 mM Tris buffer at high pH (8–9) and 0.05–0.5 SDS under gentle agitation for 5 min, then the gel is rinsed in ultrapure water. Gels can be stored indefinitely without staining loss.[33] Zinc staining is also utilized,[34] in which after electrophoresis, the gel is soaked for 30 s in ultrapure water and incubated in 0.2 M imidazole, plus 0.1% SDS with gentle agitation. The gel is then washed for a few seconds in fresh water and incubated in 0.2 M zinc sulfate for 1 min. The precipitate of zinc-imidazole-SDS on the gel surface leaves unstained protein bands. However, in these cases, chelation of metal ions with EDTA (0.25 M EDTA in Tris buffer for 30 min) is needed to elute proteins. This is a disadvantage, in that extensive EDTA destining can cause loss of protein as proteins are not permanently fixed. A sensitive, fast (completed within 1 min) and spontaneously reversible (after 30 min) staining was reported,[35] not based on metal ions. The gels are saturated in 8% (v/v) methyl trichloroacetate ester (MTA) in 38% isopropanol. After 1 h incubation, gels are put in ultrapure water and precipitation of MTA produces a negative image of proteins over the gel as opaque micelles are formed that are excluded from the protein spots.[35] However, this method is prone to small artifacts during MS identification, due to methylation of proteins by MTA.


Invisible Staining Procedures

Fluorescent Pre-electrophoretic Stains

A number of noncovalent fluorescent dyes are in use for staining of proteins either before or after electrophoresis[36] (reviewed in [16]). Their labeling of proteins can either involve a covalent bond with the protein or not. Among pre-run staining methods, lysine-binding dyes, the cyanine-based CyDyes (GE Health Care, NJ, USA) Cy2, Cy3 and Cy5[16,37] are widely used. These bind covalently with lysine residues (to a single lysine in each protein when the dye/protein ratio is 1–3%) via an amide linkage.[14] The use of three dyes allows differential images (DIGE)[11] of samples run in a single gel, which is their most interesting application as it rules out run-to-run differences in comparing proteins from two different samples. In DIGE, two samples plus one internal reference are labeled with a CyDye mixed and then scanning generates an image for each. Very sensitive cyanine dyes have been developed, that is the dyes CyDye Fluor Cy3 and Fluor Cy5 that covalently bind to the cysteine residues via a thioether bond. Labeling of cysteinesulfhydryl groups can be carried out with monobromobimane.[16] En epicocconone-based stain named 'deep purple' interacts with lysine amides.[38]

 

Fluorescent Post-electrophoretic Stains

SYPRO dyes are post-electrophoretic stains (Molecular Probes, Eugene, OR, USA). These deserve citation for their ease of use, sensitivity (1–10 ng: similar to silver-staining methods) and full compatibility with MS techniques.[7,39] In fact, SYPRO dyes do not contain interfering chemicals (such as glutaraldehyde), do not chemically modify proteins during staining and are reversible. SR, in particular, is the most used, having an excellent linear dynamic range (about four orders of magnitude) and a detection limit of around 0.5–5 ng.[7] SR binds to basic amino acids by electrostatic interaction similarly to colloidal CBB. SYPRO Tangerine can also be used in non-fixative solutions, permitting subsequent procedures.[7,39] The principal drawback of these fluorescent dyes, besides their being invisible and costly, is the need for a specific equipment to visualize them.

Experimental

In order to have a quantitative and qualitative comparative evaluation of the two methods, as it is difficult to compare gels from different papers, this section provides a comparison of two different staining methods, namely Colloidal CBB according to Pink et al. [27] and 'blue silver' according to Candiano et al. performed in blind mode. Figure 1 shows two monodimensional gels conducted using the discontinuous buffer system of Laemmli[40] of human serum albumin (HSA), loaded in sequential dilutions, starting from 100 ng and diluted by up to seven orders of dilution. Figure 1A shows the implementation of 'blue silver' staining, according to Candiano et al.:[25] after run, the gel was briefly washed with ultrapure water and then fixed in 20% methanol for 30 min. Then gel was treated with a small amount of staining solution (10% phosphoric acid, 10% ammonium sulfate, 20 % methanol, 0.12% CBB G-250) for 5 min. Then this solution was discarded and staining with the same solution was performed overnight; destaining was conducted with ultrapure water. Figure 1B represents the result of Colloidal CBB staining according to Pink et al.:[27] after run, the gel was fixed by protein fixation in 30% ethanol containing 2% phosphoric acid, the gel was then stained with 0.02% CBB-G250 in 8% (w/v) phosphoric acid, 5% aluminum sulfate and 10% ethanol for 3 h; destaining was conducted in 2% phosphoric acid and 30% ethanol for 30 min, then the gel was left in ultrapure water overnight.[27] Data in panels C and D represent the altimetry of the densitometric analysis of the gels in panels A and B, respectively. The output in terms of signal-to-noise ratio in the two staining procedures was considerably different. In fact, panel C shows that it was possible to read human serum albumin concentrations up to the 7th dilution, while in panel D only the 6th dilution was detectable. Therefore, Candiano et al. [25] has a sensitivity of 1.5 ng, higher than that of Colloidal CBB method from Pink et al. [27] Furthermore, initial background with the protocol by Pink et al. [27] was relatively higher. It may be speculated that this higher background is due to the effect of mordant, as this is the major difference between the two methods.

 

Figure 1. Comparison among two protocols for colloidal Coomassie brilliant blue staining used to stain two monodimensional SDS-PAGEs where human serum albumin was loaded in sequential dilutions, starting from 100 ng, and diluted up to seven orders of dilution. (A) The implemented 'blue silver' staining (10% phosphoric acid, 10% ammonium sulfate, 20% methanol, for 30 min 0.12% CBB G-250, after rinsing and fixation in 20% methanol, for 5 min) where methanol was added by the 'drop-by-drop' method. Destaining was conducted with ultrapure water. (B) The result of colloidal CBB staining (0.02% CBB-G250 in 8% (w/v) phosphoric acid, 5% aluminum sulfate and 10% ethanol; for 3 h, after fixation 30% ethanol, 2% phosphoric acid). Destaining was conducted in 2% phosphoric acid and 30% ethanol for 30 min, then in ultrapure water overnight. (C & D) The altimetry of the densitometric analysis of stained gels. CBB: Coomassie brilliant blue.

In time, the method by Candiano et al.,[25] confirmed its efficacy and was demonstrated to be open to a use, which the authors describe here, concerning the mode of preparation of the staining solution, rather than its recipe. The critical point lays in the mode of addition of methanol, which facilitates the staining.[24] Methanol addition to the solution of 10% phosphoric acid, and 10% ammonium sulfate is a critical step. It should be added extremely slowly (ideally in a 'drop-by-drop' manner, i.e., in small amounts and not in one step), under continuous and vigorous stirring in order to preserve the micellar colloidal suspension of the dye. This improves sensitivity (Figures 2 & 3). Indeed, the final physicochemical conditions of the solution are not the same in either case. The physical state of the solution can be easily inferred by the color of the solution: it will be a dusty greenish-blue when addition has been performed slowly but light blue if addition has been carried out incorrectly (too quickly). In the latter case, the homogenous blue color denotes dispersion of the micelles, with loss of the improved sensitivity.

 

Figure 2. Comparison among Coomassie brilliant blue-G250-based stainings of protein spots of human serum separated by in 2-DE. The first dimension was a 'Soft IPG' (24 cm nonlinear pH 3–10 Immobiline strips, IPG). Isoelectric focusing was performed at 17°C (120 kV/h). IPG were then incubated for 20 min in 0.05 M Tris-HC1, pH 6.8, 6 M urea, 30% (w/v) glycerol and 5% (w/v) SDS and embedded onto the SDS-PAGE slabs with 0.5% w/v melted agarose, plus 0.001% w/v bromophenol blue. The second dimension was a 6–16% gradient polyacrylamide gel (180 × 160 × 1.5 mm) run at 45 mA/gel. Gels were stained with the followings after electrophoresis methods: (A) 'blue silver' staining: 10% phosphoric acid, 10% ammonium sulfate, 20% methanol, for 30 min 0.12% CBB G-250, after rinsing and fixation in 20% methanol, for 5 min; (B) colloidal CBB staining 0.02% CBB-G250 in 8% (w/v) phosphoric acid, 5% aluminum sulfate and 10% ethanol; for 3 h, after fixation 30% ethanol, 2% phosphoric acid; (C) CBB staining according to Neuhoff (gel was stained in 2% phosphoric acid, 10% ammonium sulfate, 20% methanol, 0.1% CBB G-250 overnight). CBB: Coomassie brilliant blue.

Figure 2 shows three 2-DE of human serum as stained with the method 'blue silver' staining according to Candiano et al. [25] (panel A); colloidal CBB staining according to Pink et al. [27] (already detailed) (Panel B) and CBB staining according to Neuhoff[24] (gel was stained in 2% phosphoric acid, 10% ammonium sulfate, 20% methanol and 0.1% CBB G-250 overnight) (Panel C). For the first dimension of the 2-DE, 'Soft IPG' (24 cm nonlinear pH 3–10 Immobiline™ strips (GE Healthcare, NJ, USA), IPG) were employed, as previously described.[41] The sample was loaded in at the basic end of the rehydrated IPG. Isoelectric focusing was performed at 17°C by progressively increasing the voltage (100 V for 2 h, up to 8000 V, for a total of 120 kV/h). IPG were then incubated for 20 min in 0.05 M Tris-HC1,pH 6.8, 6 M urea, 30% (w/v) glycerol and 5% (w/v) SDS, then embedded onto the SDS-PAGE slabs with 0.5% w/v melted agarose, containing 0.001% w/v bromophenol blue. The second dimension was a 6–16% gradient polyacrylamide gel (180 × 160 × 1.5 mm) run in a Protean® II Multi-Cell vertical chamber (Bio-Rad, CA, USA), at 45 mA/gel. Picking of spots (1 mm diameter) was performed by a SpotCutter® (Bio-Rad, CA, USA). These were trypsin digested and subjected to MS analysis using an LTQ™ linear trap mass spectrometer (Thermo Electron, CA, USA) equipped with a Jupiter® C18 column 250 × 1 mm (Phenomenex, CA, USA). Spectra were acquired in automated MS/MS mode. Peptides were eluted by an acetonitrile gradient. Sequence coverage in the three 2-DE was very similar (between 20 and 22%). In fact 100 ng of protein had been loaded in all cases. The difference lays in the absolute possibility of spot detection, in that a better stained spot will have a higher probability to be detected. Therefore, the use of a sensitive staining allows to increase the number of identified spots.

Figure 3 shows the digitized image of a classical 2-DE performed on normal human urine sample processed with 'proteomineering' combinatorial peptide ligand libraries protocol,[42,43] stained with the present implementation of 'blue silver' protocol. A total of about 2000 spots were detected. When the 'blue silver' protocol is performed as detailed above, it gives an optimal signal-to-background ratio, good to optimize the dynamic range.