This chapter describes electrophoretic-based protein assays, focusing on the current methods for total protein assay, post-translational modification (PTM) assay for common proteins, phosphorylation assay, and glycosylation assay. In the 20 years since the first publication of this volume, the SDS-PAGE and subsequent protein stains have remained an important laboratory procedure for determining the composition and characteristics of protein-containing mixtures at all stages of purification protocols. The conventional approach to protein profiling involves sampling at each step of purification, from cell lysate to final product, to evaluating peptide composition, purity, and quantitation. Therefore, the samples taken at each step should progressively provide a more concise peptide profile, thus showing a gradual increase in the percentage of target proteins.
Authors: Burgess et al., Translator: Chen Wei, this experiment is from the "Guide to Protein Purification".
Operation method
Protein gel staining assay Move Detection of total protein 1. Total protein colorimetric staining The simplicity of visual detection, relative ease of use, and a broad base of users familiar with the method have made Caulmers Brilliant Blue (C B B )-still the most commonly used gel stain for total proteins. If a higher sensitivity of detection is required than with Kaumas Brilliant Blue staining, then silver staining is the colorimetric method of choice. If mass spectrometry analysis of cut proteins is required, then staining methods that do not introduce covalent protein modifications are preferred. The simplicity of visual detection, relative ease of use, and a broad base of users familiar with the method have made Caulmers Brilliant Blue (C B B )-still the most commonly used gel stain for total proteins. If a higher sensitivity of detection is required than with Kaumas Brilliant Blue staining, then silver staining is the colorimetric method of choice. If mass spectrometry analysis of cut proteins is required, then staining methods that do not introduce covalent protein modifications are preferred. Kaumas Brilliant Blue Staining Kaumas Brilliant Blue R-250 and its dimethyl derivative Kaumas Brilliant Blue 0 2 5 0 are triphenylmethayaki di-transverse acid dyes that stain protein bands a bright blue color. The bands interact electrostatically with protonated basic amino acids (lysine, arginine and histidine) and are hydrophobically bound to aromatic residues by hydrophobic interactions. The dye does not bind with high affinity to the poly(acrylamide) but penetrates the gel medium and binds with low affinity to it, thus requiring a decolorization step unless the dye solution is a colloidal formulation. Typically, Thomas Brilliant Blue is used as the endpoint stain, which provides the fixation and staining steps. Staining with Kaumas Brilliant Blue R-250 is accomplished under acidic ethanol conditions with a dosage of Kaumas Brilliant Blue R -250 Upon complete analysis of the Caulophylline blue stain, it is known that colloidal formulations are not conducive to efficient entry of the stain into the colloidal medium where it is specifically bound to the protein bands, resulting in a reliable quantification and a sensitivity that is closer to the standard Caulophylline blue R-250 stain as described above, allowing for staining with a low background. When used as a colloidal stain, Phosphorothioate 250 is preferred over Phosphorothioate R-250, and the initial formulation consists of 0.1% Phosphorothioate g _250 solubilized in 2% (?w/v) of a solution of Phosphorothioate g _250, which is then dissolved in 2% (?w/v). Most commercially available formulations of komas bright blue dye are derived from the various formulations and solutions described above. There are numerous recommendations for fixation, after electrophoresis the gel is washed with water to remove most of the S D S from the dye solution, the washing time can be from a few hours to overnight, this simple procedure helps to observe good results. Protein bands can be observed within a few minutes, with the background initially clearing and eventually turning light blue. After staining, the gel is washed with water to minimize the background. There is a recent summary of simple and practical Cauloblue staining protocols, including a "hot" Cauloblue staining protocol, i.e., Cauloblue R-250 dye formulation based on acetic acid at 90°C, or a "fast" 5(T C) Cauloblue ○-250 colloidal dye based on trichloroacetic acid. Programs. Commercially available. Silver Dyeing It is widely accepted that silver staining is the standard for all other ultrasensitive staining methods, and is the most complex and variable protein gel staining methodology, with dozens of published protocols, all of which require a number of stepped steps; protein detection is based on the reduction of silver ions bound to proteins to metallic silver or, as occurs less frequently in some protocols, results in a localized silver sulfide precipitation. Silver staining can be used as a rigorous standard for limiting the sensitivity of the assay, and quantification is not a simple task; it depends on the nature of the complexes in the chromogenic step and the differences in the sensitivity of the silver signals between different proteins. There are commercially available silver-staining kits that use a variety of well-tested methods from the literature. The key steps are usually described below. (1) Fix the gel to immobilize the protein bands and remove interfering substances from the gel, such as S D S, buffer, and salt, which bind silver and cause background staining. (2) Incubate the gel with a substance that binds the protein and increases the silver binding capacity, or that interferes with the background staining caused by the remaining unbound silver. In short, these various methods are related to sensitization. (3) Silver ion immersion treatment of gels. (4) The bound silver ions are reduced to insoluble and visible metallic silver, and a colorful silver-stained signal appears. (5) The color development process is terminated to prevent background staining in the gel medium from obscuring the protein bands. The time dependence of the last two steps leads to the complexity of the technique, as do several time-consuming washing steps in steps (2) to (4). The silver staining method is clearly differentiated on the basis of the silver reagent and the corresponding development conditions. The alkaline method uses a silver diamine complex in an alkaline environment as the silver reagent and develops in an acidic formaldehyde solution, while the acidic method uses weakly acidic silver nitrate as the silver reagent and develops in an alkaline formic acid solution. Acid staining has recently become more popular because it reduces costs relative to alkaline methods and the background staining is easier to control. A number of sensitizers are currently available. Aromatic transverse salts (e.g., disulfonated naphthalene or sulfosalicylic acid) bind proteins as well as silver; the remaining protein-bound S D S also enhances silver binding. In the same way, the intensity of silver staining can be improved if a gel that has been stained with Caulophylline blue is silver-stained again. Glutaraldehyde has been widely used as a sensitizer, the principle of which is the introduction of a reducing aldehyde group, which can combine with the free amino group in proteins. Sulfide reagents, such as tetrathionate or thiosulfate, can introduce free S2- ions in the vicinity of proteins, which immediately react with silver ions and form insoluble silver sulfide. Because the use of glutaraldehyde during fixation and sensitization results in protein modification, mass spectrometry analysis of proteins after silver staining can be problematic. Methods that are compatible with mass spectrometry do not use glutaraldehyde, which may result in relatively low sensitivity. If the sensitization step relies on tetrathionate and thiosulfate, the development step will be correspondingly longer. Therefore, if mass spectrometry is required after silver staining, the colloid can be stained with Kaumas Brilliant Blue and then silver stained without glutaraldehyde, which is a convenient two-step chromatographic method to estimate the purity of a protein sample. Zn 2+ counterstaining After S D S -P A G E , there are a number of methods for rapid staining of proteins using metal cations without relying on fixatives or organic dyes. Zn 2+ counterstaining utilizes the binding of proteins and protein-SDS complexes and the isolation of Zn 2+ in an environment where the precipitation reaction between imidazole and Zn2+ produces zinc imidazole (Zn Im 2) and an opaque background that contrasts with the transparent protein-SDS Zn 2+ regions. The technique is a non-fixed step, useful for subsequent microanalysis in anticipation, such as bioanalysis of elution bands or enzymatic analysis, and is compatible with mass spectrometry or microsequencing methods. The stain is fast and rapid, but the sensitivity of the assay is lower than that of the Khmer Rouge stain. The procedure for rapid counterstaining of Zn 2+ on gels of I m m thickness is described below. (1) After electrophoresis, the gel was immersed in ○.2 m o l /L imidazole, 0.1% S D S for 15 m i n . (2) Discard the imidazole solution, and then incubate the gel in 0.3 m o l /L zinc sulfate for 30 to 45 s to develop the image. (3) Discard the above development solution, and wash the gel with water several times, each time i m i n . (4) Store the gel in 0.5 % (m/V) sodium carbonate. (4) Store the gel in 0.5 % (m/V) sodium carbonate. Overdevelopment can be a problem, but it can be solved by soaking the gel in 100 m ml/L Glycine to dissolve the excess ZnI2. Although the transparent protein bands in an opaque background do not have as much color contrast as the Caumas Brilliant Blue or Silver stains, it is possible to illuminate the gel image against a dark background (Fernandez-Patron, 2005; see also Fernandez-Patron, 2005; Fernandez-Patron, 2005; Fernandez-Patron, 2005). Fluorescent staining combines assay sensitivity (comparable to silver staining) with the simplicity of the staining process (as with the Cauloblue stain or Zn2+ counterstaining) and a linear quantitative range 10-100 times greater than that of colorimetric methods. The assay is instrument dependent and requires a monochromatic excitation light source, selective optical filters that separate the longer wavelength emitted light from the shorter (and brighter) excitation light, and a detection module. For many fluorescent dyes, visual detection is possible, but the flexibility is limited. Fluorescent dyes are generally categorized into two groups: fluorescent dyes that exhibit a significant enhancement of fluorescence at the site of protein bands and autofluorescent dyes that specifically bind to protein bands without binding to the gel matrix. The first commercialized total protein fluorescent dyes, S Y P R O O r a n g e a n d S Y P R O R e d gel dyes appeared in the 1990s. These dyes were initially used for routine fluorescence detection of stained DNA in gels by transmission of 300 nm ultraviolet light followed by Polaroid photography. The development of these commercial fluorochromes was aimed at establishing a simple, one-step workflow for specific staining and documentation of total proteins similar to that of ethidium bromide or SYBR Greene DNA staining, with detection sensitivity that exceeds that of the Khmer Rouge staining method. Further development of total protein dyes has produced a number of commercial products or formulations with assay sensitivity ranges that approximate or even exceed those of the silver staining method. Those fluorescent gel staining methods that do not require covalent labeling of the sample prior to electrophoresis are compatible with mass spectrometry analysis that elutes the protein bands later. Nile Red Protein Gel Stain Nile red is a phenanthrozine ketone dye that shows strong fluorescence enhancement when transferred from water to hydrophobic environments such as SDS particles or protein-SDS complexes. Nile Red Does Not Interact Significantly with S D S Monomers" This feature was utilized to develop a rapid total protein staining method without fixation suitable for S D S gels. The protocol requires that electrophoresis be performed under non-standard conditions, i.e., an electrophoresis buffer S D S content of 0. 0 5 % (m / V ) below the critical micellar concentration of the decontaminant, rather than the 0. 1 % (m / v ) S D S content normally applied to 1D and 2D S D S -P A G E . Protein samples should be prepared to specification (Garn n , 1990a; 1990b) so that the SDS-protein complexes remain stable during electrophoresis and the migration of the protein bands is considered normal. The method is simple and rapid: Nile Red stock solution (0.4 m g/m L in DMSO) is diluted 200-fold in water to a final concentration of 2 ug/m L. More than 10 times the volume of the gel is added to each gel (e.g., 50 m L of dye is required for a 5 m L minigel), which is immediately swirled thoroughly. The dye settles quickly in water, so the ideal time for staining should be 2 to 5 m i n , beyond which the effect will not be enhanced. After staining, wash the gel briefly with water. The gel can be excited with UV or green light, and the protein bands will appear reddish in color. The sensitivity of the assay is similar to that of the Caulophylline blue stain. Because of the lack of fixation, Nile red-stained gels have better transfer efficiency for subsequent electroswimming trace transfer (Daban, 2001; Daban et al., 1991). Precipitation of the dye creates a highly fluorescent background in the gel medium, and the affinity of the dye for the S D S colloid coupled with the insolubility of the dye makes this method unsuitable for use with S D S ^ P A G E containing 0-1 % S D S in the electrophoresis buffer. The photostability of the dye is also problematic. S Y P R O O r a n g e , S Y P R ○ R e d and S Y P R O S Y P R O R e d and S Y P R O O r a n g e protein gel stains were described by Steinberg et al. (1996a ;1996b ) and H a u g l a n d et al. (I" 7), and S Y P R O T a n g e r m e protein gel stains were described by Steinberg et al. (2000b ) and Y u e et al. ( 2003), and all three stains were similarly discussed by Steinberg et al. These stains contain a hydrophilic functional group, an aromatic fluorophore, and an aliphatic tail, giving the dyes both good water solubility and a strong ability to intercalate into protein-SDS complexes, SDS particles, or membranes, with strong fluorescence enhancement in nonpolar environments. These characteristics, together with good chemical and optical stability, allow the dyes to be generalized to standard buffer conditions (○. 1 % SDS) SDS in a variety of easy staining steps. 1 % S D S )S D S --P A G E after staining with a sensitivity of detection that exceeds that of colloidal Khao Ming Blue 0 2 5 0 dyes. Non-standard buffer conditions (0.05% S D S ) , these S Y P R O O r a n g e a n d S Y P R O R e d are patented sulfopropylaminostyryl dyes, commercially available in a 10 m m o l /L stock solution dissolved in DMSO. The staining method is simple: (i) after SDS-PAGE, the gel is placed in the staining solution; (ii) before image acquisition, the gel is washed briefly with water. To prepare the staining solution, dilute the dye 5,000 times to 2 umol/L in acetic acid [7 % (V/V) is standard, 2 % to 10 % are equally effective]. The staining solution is usually prepared ready to use, but it can be stabilized for several months. After electrophoresis, the gel is placed in 10 to 20 times the gel volume (50 to 100 mL for a 5 mL microgel) of the staining solution, which is placed in a polypropylene or polycarbonate dish and stirred gently and continuously. The dish of staining solution containing the gel is placed in an ultraviolet light box or in a polypropylene or polycarbonate dish with constant gentle agitation. S Y P R O T a n g e r i n e Protein Gel Dye is a patented carbazolylvinyl dye that can be distinguished from S Y P R O O R a n g e and S Y P R O R e d protein gel stains based on the recommended dilution of the dye and the intended use. This protein dye can be used in neutral p H buffer (50 m m o l /L squamate, 150 m m o l , 'L N a C l ,p H 7. 0), when the protein bands are not immobilized and can be subsequently enzymatically spectrometrically analyzed, eluted, and replicated for use in analyzing their activity in vitro or for electrotransfer blotting. The dye is commercially available as a 10 m m o l /L reservoir; the dye is diluted 5000-fold to 2 u m m o l /L and dispensed ready to use. The staining process is identical to that of S Y P R O O r a n g e , and can be completed within I h; the gel is washed with water prior to image acquisition. Spectral excitation is accomplished using either a UV light source or a blue light source. SYPRO Ruby Protein Gel Stain and other ruthenium-based formulations Organometallic ruthenium ion-based luminescent stains for the detection of proteins in gels or on blots, such as S Y P R O R u b y Protein Gel Stain, which can provide fluorescence detection sensitivity comparable to, or even exceeding, that of silver staining in a simple end-point staining step, have also been introduced into the S D S--P A G E after (Berggren et al., 2000) or after isoelectric focusing electrophoresis (Steinberg et al., 2000a) in ready-to-use stain formulations. The development and formulation of these dyes is based on colloidal Kaumas Brilliant Blue staining schemes in which the organic component chelates luminescent ruthenium (II) and provides the basis for non-covalent proteins in a similar manner to the Kaumas Brilliant Blue staining, i.e., firstly ionic interactions with the basic amino acids and secondly hydrophobic interactions. （Ruthenium (4,7-diphenyl-1,10-phenanthroline)ruthenium [ruthenium II tris (1) Fix the gel with 50 % (V/V) methanol, 10 % 0 VV ) acetic acid for 30 min to overnight. (2) The gel was stained with soap ruby dye. This is a final coloration method, which is stable for 3 h until overnight. (3) Simple gel decolorization was carried out with 10% (〇 //V) methanol, 7% ( V /V) acetic acid. The staining process can be accelerated by utilizing a microwave oven based protocol. SYPRO R uby protein gel dyes have relatively high extinction coefficients and quantum yields, making them very bright, chemically stable and photostable. Spectral excitation can be achieved with either UV or blue light; the protein bands appear visually orange-red in color. The mechanism of fluorescent protein staining is the differential binding of the dye to the protein bands and to the gel medium, which does not bind the dye. The gel medium does not bind to the dye, so the protein stain itself is not fluorescent. The brightness, stability, sensitivity and ease of use of SYPRO R uby protein gel dyes, coupled with the extensive literature on their proteomic applications and an aggressive marketing campaign, have led to the development of this product. Epicocconone Protein Gel Stains: Deep Purple and Lighting Fast Protein Gel Stains Epicocconone Protein Gel Stain is a fluorophore derived from the fungus EpicoccMOT Tiignwn and is used in total protein staining kits. The dye has been used under various trade names, including DeepPurple and Lighting Fast Total Protein Gel Stain, in a variety of protocols. The following is one option. (1) The SDS-PAGE gel is solidified in 7.5% (V/V) acetic acid for 1h. (2) Wash the gel with water (2X 30 m i n ). (3) Dilute the fluorophore stock solution in aqueous solution, and then stain the gel in this aqueous solution for I h. The gel is then stained with the fluorophore stock solution. (4) Incubate the gel in 0. 05% (V /V ) ammonia (3X 10 m i n ). (5) Immediately capture the image, which can be excited with ultraviolet light, visible blue light, or visible green light. The sensitivity of the stain is comparable to or better than that of the S Y P R O R u b y protein gel dye (Belland K a r u s o , 2003; Mackintosh etal., 20 0 3 ) . The staining is reversible and compatible with mass spectrometry analysis. The dark red signal is not particularly significant and its sensitivity depends entirely on the fluorescent properties of the stain, resulting in a weak background. The staining method appears to require trace amounts of S D S that remain complexed with the protein after acetic acid fixation. The stain fluoresces only weakly (green) in water, but in the presence of SDS-treated proteins, the fluorescence is enhanced and red-shifted. Epicocconone protein gel dye has been shown to react with lysine to form a fluorescent adduct that can be hydrolyzed by alkali, thus producing a burst of light and making the staining reversible (Coghlan et al., 2005). Concentrated staining reserves must be kept frozen and thawed before use. Image capture should be performed immediately after staining is completed, taking into account stability factors. Fluorescein Derivatives Fluorescein derivatives containing hydrocarbon tails are very effective protein gel stains. The use of 5-dodecanoylamino-(C 12-F L ), 5-hexadecanoylamino-(C 16-F L ), and 5-octadecanoylamino-eicosanoid-(C 18-F L ) in staining protocols for a number of samples analogous to the classical Kh Khao Maas Brilliant Blue R-250 stain proved to be comparable to the sensitivity of the silver stain. The most effective fluorescent group is C 16-F L. For example, the gel is stained with 30% (V/V) ethanol, 7.5% (v/v) acetic acid, 1 umol/L dye, and the solution is changed twice. The dye is then washed with water, which is also changed twice, and finally decolorized with 7.5% acetic acid. The basic principle of the incendiary nature of the dye is known to be binding to residual SDS bound to the complex proteins; this is compatible with mass spectrometry (Kager et al., 2003). The spectral excitation and emission of fluorescein is accomplished by blue and green light, respectively. K r y p t o n protein gel stains KRPON Protein Gel Stain is a patent-protected formulation of a hydroxyquinoline-based dye with a green excitation spectrum and an orange burst color (Wolf et al., 2007). Krypton Infrared Protein Gel F l a m i n g o Protein Gel Stain Flamingo Protein Gel Stain is a patent-protected formulation of a coumarin-based anthocyanine dye (Berkelm a n , 2006). This staining protocol requires fixation to be completed prior to staining. The dye can be purchased as a stock solution of I O X and diluted with water prior to use. The dye exhibits fluorescence enhancement when bound to proteins. The stain has a weak background, and a short decolorization step has been reported to further diminish the background. The excitation spectrum is in the green range with an orange/red signal. The stain is considered comparable to silver and S Y P R O R u b y and is also compatible with mass spectrometry. L U C Y Protein Gel Stain LUCY Protein Gel Stain is a patent-protected formulation of trimethinecyanine dye that exhibits fluorescence enhancement in SDS/protein mixtures. Staining is usually performed in diluted acetic acid in the same manner as S Y P R O O r a n g e - . The stain is also reported to be compatible with mass spectrometry (Kovalska et al., 2006). The development and use of succinic ugly imide ester, a charge-balanced cyanine dye (Cydye) for covalent amino labeling of protein samples prior to electrophoresis, has served as the basis for fluorescent two-dimensional difference gel electrophoresis (2- D D I G E , - a very important proteomic For more product details, please visit Aladdin Scientific website
important flexibility in the timing of the fixation and staining steps. The Caulmers Blue stain is non-covalent and reversible and does not interfere with subsequent mass spectrometric analysis of the cut+ protein bands.
0.025% to 0.10% (m/V dye solution), 30% to 50% ( V/V) methanol (or ethanol, not as common as methanol) with 7 % to 1 0 % ( V/V) acetic acid solution. After the dye is dissolved, the solution is filtered through W hatman No. 1 filter paper to produce a stable composition. Fixation and coloration are usually accomplished in a solution of approximately 10 times the gel volume, such as the standard microgel which requires 50 mL. In pursuit of the most sensitive and always consistent results, fixation and staining of gels need to be performed in the same acetic acid solution. Under these conditions, the gel shrinks. Decolorization in 7 % acetic acid and reduction of ethanol can restore the gel to its full size. Decolorization usually requires several solution changes and can be accelerated by the addition of dye absorbent paper or foam rubber/plastic.
The initial formulation consisted of dissolving 2% (?w/v) of Caulophyllum Blue g _250 in 2% (?w/v) of pitric acid and 6% (n/v) of ammonium sulphate (Neuhoff et al., 1985). An improved and widely used formula is 10% (?WV) ammonium sulphate dissolved in 2% (?WV) phosphoric acid, followed by the addition of 5 % (?m/V) aqueous solution of a komassie blue 0 2550 stock to a final concentration of 0.l % (?w/V); methanol should be added to a final concentration of 20% (?V/V) before staining (N e u h o f fet al., 1988). The addition of methanol increases the proportion of monodisperse dyes, resulting in faster staining and more visible bands, but some background colors also become visible; increasing the concentration of ammonium sulfate drives the dye into a colloidal state. In addition, the updated formulation suggests increasing the dye and phosphoric acid concentrations, i.e., the final formulation should contain 0.12% (m/V) dye, 10 % (OT/V) ammonium sulfate, 10 % (m/V) phosphoric acid, and 20 % (m/V) phosphoric acid. V) phosphoric acid and 20% (V/V) methanol, all of which form a single stable solution. To prepare the solution, the above amounts of phosphoric acid, ammonium sulfate and powdered dye are added sequentially to water to 80% of the final volume, and then methanol is added to the final volume (Candiano et al., 2004). The colloidal dye solution cannot be filtered.
Some protocols also include the use of simple microwave-based protocols to accelerate the dyeing (and decolorization) of Phosphor Blue 0250 Colloidal Dye.
validated protocols and reagent mixtures that provide reproducible results for first-time users. It is also an important learning and benchmarking tool for those who want to improve and optimize 'home-made' silver staining reagents and protocols for routine in-house use. Merril (1990), R a b i U o u d (1990) and P o l a n d et al. (2005) revised and summarized the original silver staining method
Merril (1990), Rabiuud (1990) and Pollán et al.
Fernandez-Patron et al., 1998).
For many fluorescent dyes, they can be detected visually, but this is not as sensitive as using a camera or instrumentation. Any fluorescent dye will exhibit some degree of photobleaching as a result of exposure. Many commercially available fluorescent dyes have been improved and are relatively light stable. Nevertheless, care should be taken to avoid prolonged exposure of the gel to bright external light prior to visual inspection and image acquisition. The excitation and emission limits for the fluorescent stains and dyes discussed in this chapter are presented in Table 31.1. In general, the colors of light excitation and emission are often distinguished by broad categories, i.e., ultraviolet (UV) 250 to 400 nm; blue 400 to 500 nm; green 500 to 550 nm; yellow/orange 550 to 580 nm; red 580 to 650 nm; and near-infrared 650 to 850 nm. This is also the purpose of the following discussion.
Tangerine Protein Gel Stain
Under non-standard buffer conditions (0.05% S D S ), these dyes provide a 4-fold increase in staining sensitivity compared to Nile Red, but this is not necessary for their use.
Staining can be monitored periodically by placing the gel-filled dish in a UV light box or blue light transilluminator, or by using a hand-held UV light or blue light LED. Typically, for 10 ug of cell lysate, a large number of fluorescent bands of protein can be seen within 10 m i n or 15 m i n against a fluorescent background. As the staining process proceeds, the signal of the protein bands increases, and the bands of less abundant proteins become more pronounced against the attenuated background. For gels as thick as I m m, staining can be completed within I h, and the gel will not show up in the staining solution.
was stable for several days in the staining solution. The protein-SDS-dye complex is stable in diluted acetic acid, but the remaining SDS will diffuse out of the gel, causing a weak background. Therefore, decolorization should be minimized. Prior to image acquisition, the gel needs to be washed with water to remove the remaining dye, SDS, and acetic acid from the gel surface, and the water needs to be changed twice during the wash, with each wash requiring 2 to 3 m i n .
(bathophenanthroline disulfonate)] The detailed description of the preparation, the staining scheme and the comparison with the Sampler's protein gel staining method show that the staining principle of Sampler's method is very similar to that of ruthenium staining; the mass spectrometry analysis reflects some minor differences between the two methods, which are attributed to their proprietary chemical properties. Mass spectrometry revealed some minor differences between the two, which were due to their proprietary chemical properties. Subsequently S Y P R O
R u b y gel stain was improved, and the performance of the original formulation was enhanced to a ready-to-use stain for S D S - P A G E and isoelectric focusing gels (Berggren et al, , 2002). Thus, S Y P R O R u b y differs from ruthenium staining not only in the synchrotron chemistry, but more importantly in the formulation of the stain, i.e., it has a stable, ready-to-use, and a stable, easy-to-use staining solution.
It has a stable, ready-to-use staining solution and a relatively simple staining protocol.
provided the standard of comparison for subsequent development of protein gel stains, just as silver staining remains the benchmark for assay sensitivity.
A dye containing patented coumarin/fluoroquinolone and/or Martina-based dyes with a red/near-infrared excitation spectrum and a near-infrared emission spectrum (Czemely et al., 2008). Components of these products are available in 10X form and need to be diluted to a usable solution. These products are comparable in sensitivity of detection and range of dynamic linearity to the Sample Roruby and DePrint Protein Gel Stain. The staining protocol follows the standard fixation-staining-rapid destaining workflow and has been improved for rapid staining, so that the entire staining process can be completed in 1 to 4 h. For maximum sensitivity and signal linearity, the protocol can be extended. These stains are compatible with mass spectrometry.