Dye Hard: Protein Gel Staining Products

he search for methods to visualize proteins resolved by electrophoresis on everything from cellulose acetate strips to polyacrylamide gels goes back to the origins of the electrophoresis itself. When DISC electrophoresis was introduced in the early 1960's (B.J. Davis, Annals of the New York Academy of Science, 121:404, 1964), amido black was the protein stain used most frequently. As the need for staining methods with increased sensitivity and staining uniformity grew to meet the demands of protein quantitation in gels, amido black faltered. The dye lacked the necessary sensitivity, suffered from lot-to-lot variations, and often stained proteins multichromatically (C.M. Wilson, Methods in Enzymology, 91:236-47, 1983). A combination of some clever ideas and trial and error over the years has since given rise to a collection of highly sensitive and effective protein gel staining techniques and products. These include the popular and reliable Coomassie Blue, a variety of silver staining techniques, the reversible, negative staining of gels with heavy metal salts, fluorescent dyes, and three lesser-known dyes with some interesting properties. Additionally, the use of colloidal gold sols to stain proteins on transfer membranes is well characterized and very useful. Finally, an automated gel staining machine is now on the market. Silver staining is currently regarded as the most sensitive staining method, detecting as little as 1 nanogram of protein per band, compared to the 30 nanograms to 100 nanograms per band detected by Coomassie Blue staining methods (T.H. Steinberg et al., Analytical Biochemistry, 239:223-97, 1996). Proteins, however, can and do respond differently to silver staining. In fact, some proteins may not stain at all, so sensitivity values for silver staining may vary depending on the protein. Silver staining also has applications for staining DNA and RNA resolved on acrylamide and agarose gels. Here, then, is a closer look at the protein gel staining products currently available. Motivated by the lack of a suitable staining procedure for visualizing proteins separated by electrophoresis on cellulose acetate strips, Fazekas de St. Groth et al. ( Biochimica Biophysica Acta, 71:377-91, 1963) systematically tested a good number of standard dyeing techniques used in the textile industry for their suitability as protein stains. In 1963 they introduced the acid wool dye Coomassie Brilliant Blue 250 as a suitable stain because of its great sensitivity, color intensity, and the position of its absorbance peak. In 1965, Meyer and Lambert (T.S. Meyer, B.L. Lambert, Biochim. Biophys. Acta, 107:144-45, 1965) successfully applied the Coomassie Blue staining method of de St. Groth to the detection of parotid saliva proteins separated by electrophoresis on acrylamide gel strips. Their success led them to make the prophetic suggestion that "Coomassie brilliant blue could find application in investigations of other biological fluids by acrylamide-gel electrophoresis." The rest, of course, is history. Coomassie Blue was indeed originally manufactured as an acid wool dye, or a dye that stains wool under acidic conditions (C.M. Wilson, Methods in Enzymology, 91:236-47, 1983). Two variants of the dye exist. Known as Coomassie Blue G-250 and Coomassie Blue R-250, the dyes stain with green and reddish tints, respectively. The "250" is a dye strength indicator used by the original manufacturer, Imperial Chemical Industries (ICI). While ICI no longer manufactures Coomassie Blue, it still owns the trademark. PlusOne Silver Stain from Amersham Pharmacia Biotech Coomassie Stain from Bio-Rad Silver Stain from Bio-Rad SYPRO Orange from Bio-Rad The conventional Coomassie Blue/acetic acid/methanol staining procedure (F. M. Ausubel et al., Current Protocols in Molecular Biology, Unit 10.6) for acrylamide gels has seen widespread use throughout the years, but the strong background staining and resulting destaining steps needed often are inconvenient. In 1985, V. Neuhoff reported a sensitive protein stain for acrylamide gels based on the colloidal properties of Coomassie Brilliant Blue G-250 (V. Neuhoff et al., Electrophoresis, 6:427-48, 1985). This method produced stained protein bands on a clear background in less than an hour and had sensitivity levels similar to silver staining. Neuhoff's team improved this technique in 1988 ( Electrophoresis, 9:255-62, 1988). The addition of methanol and ammonium sulfate to a solution of Coomassie Blue G-250 in dilute phosphoric acid shifted the dye into its colloidal form. During staining, an equilibrium exists between the colloidal and molecular dispersed forms of the dye. The dispersed form enters the gel matrix and stains the proteins preferentially while the colloidal form is excluded, avoiding background staining. The colloidal properties of Coomassie Blue G-250, as well as the conventional staining methods, have been used by protein staining kit manufacturers in the development of numerous products. Several products are available, and they are briefly described below. Amersham Pharmacia Biotech PlusOne Coomassie Blue $34.00/40 tablets R-350 Tablets PhastGel Blue R 50-100 ng/band $34.00/40 tablets These tablets are in a readily soluble, premeasured format for preparing Coomassie Blue R-350 solutions. The company has found that Coomassie Blue R-350 is more sensitive than the more common R-250 and R-150 stains. PhastGel Blue R tablets are made specifically for use with Amersham Pharmacia's PhastGel Automated Electrophoresis System. AMRESCO Quick Coomassie Staining System 50 ng of protein $35 AMRESCO's Quick Coomassie Staining System is a two-reagent system that lets users achieve 10 minute staining and 40 minute destaining of SDS-PAGE gels. The high sensitivity of this system is due to the exceptionally low background staining. Bio-Rad Laboratories Coomassie Brilliant Blue Stain & Destain Kit 40 ng/band $90.00 Coomassie Stain $40.00 Consisting of premixed Coomassie Brilliant Blue R-250 stain and destaining solutions, this kit makes the classical Coomassie staining method easier and more convenient than ever by eliminating the need to make solutions from scratch. Only Bio-Rad offers this long time standard formulation. Bands are deep purple/blue on a clear background. This product can be used to stain SDS PAGE, native PAGE and membranes, and can be reused three to four times. The one liter of stain and the two, one liter destain solutions are ready-to-use. Biotech Holdings Stain-Destain-Dry Kit Nanogram amts. $45.00 As a company with strong ties to the educational market, Biotech Holdings has developed a less hazardous Coomassie staining method that avoids the use of methanol, yet is ten times more sensitive than the conventional methanol/acetic acid procedure. Methanol toxicity includes symptoms like fatigue, headache, visual impairment, and even death. This kit makes gel staining a much safer and environmentally friendly activity. The kit stains 20, 16 cm x 18 cm gels, or 50 mini gels. Biotech Holdings also prepares custom staining solutions and reagents. Diversified Biotech Rapid Coomassie Stain <= 30 ng/band $35.00 Express Coomassie Stain R-250 & G-250 15 ng/band $65.00 Diversified Biotech's pair of Coomassie-based staining products produces detectable bands in under an hour with little or no background. Bands are visualized between 20 minutes and 40 minutes after a 10 minute fixation step with Rapid Coomassie Stain. The Express Coomassie Stain is a colloidal preparation of either Coomassie Blue R-250 or G-250. Bands are visible within 50 minutes with this kit. The Rapid Coomassie Kit makes 1 liter of working solution, and the Express Coomassie Stain is sufficient for staining 25 mini gels. ESA, Inc. Fast Blue Colloidal Coomassie Blue Stain $30.00 ESA's Fast Blue Colloidal Coomassie Blue Stain is compatible with SDS and native PAGE gels. The easily prepared Fast Blue staining solution develops bands in as few as 30 minutes with no destaining. The Fast Blue concentrate is sufficient for staining five mini gels. Geno Technology, Inc. RAPIDstain 10 ng/band $50.00 RAPIDstain is based on the colloidal properties of Coomassie Blue, and enables band visualization in as little as 2 minutes. Mini gels are usually stained in 10 minutes, and destaining is not required. This kit will process 40 mini gels. ICN Biomedicals Coomassie Colloidal Image Enhancer <10 ng/band $42.00 Consisting of a colloidal suspension of Coomassie Blue G-250, the Coomassie Colloidal Image Enhancer intensifies the images of proteins, peptides and DNA by staining them in an enhanced dark blue color. Sold in a 500 ml volume, this product is as sensitive as most silver stains. Life Technologies BluPrint Fast-PAGE Stain 15 ng/band $94.00 Employing a novel Coomassie Blue-based staining solution that reduces background staining, the BluPrint Fast-PAGE Stain uses a simple three step method for visualizing proteins in less than an hour. BluPrint works with most PAGE and SDS-PAGE applications that use Tris-glycine buffer systems. Novex Colloidal Blue Stain Kit 10 ng/band $99.00 Based on the colloidal properties of Coomassie Blue G-250, this Novex kit produces visible protein bands in less than an hour with minimal background staining. Using only water as a destain, water-clear backgrounds are produced overnight. The Colloidal Blue Stain Kit will stain 25 gels. Owl Separation Systems Pro-Blue(TM) 10 ng/band $99.50 Pro-Blue is a colloidal blue stain that is claimed to be two to five times more sensitive than conventional Coomassie Blue stains. Used in conjunction with TCA fixation, Pro-Blue stains low molecular weight proteins frequently lost in standard methanol/acetic acid techniques. One kit stains 25 gels using 100 ml per gel. Pierce Chemical Company GelCode Blue Stain Reagent >= 8 ng/band $29.00 A recent addition to the protein gel staining products, GelCode Blue Stain reagent uses the colloidal properties of Coomassie G-250. A Water Wash Enhancement (TM) step further enhances the staining sensitivity of GelCode Blue Stain and produces a clear background. GelCode Blue works with SDS-PAGE, native PAGE, and PVDF membranes. This product will stain 25 mini gels for an economical $29.00. Roche Molecular Biochemicals (formerly Boehringer Mannheim) BM Fast Stain < 10 ng/band $30.00 Roche Molecular Biochemical's BM Fast Stain is a one hour to two hour staining procedure based on a modified colloidal Coomassie staining method. The technique is suitable for staining proteins in polyacrylamide gels after electrophoretic separation under reducing or non-reducing conditions in IEF, SDS, or native gels. This product stains 25 mini gels and contains no hazardous chemicals. CompanyPhoneWeb or e-mail Accurate Chemical & Scientific Corp. 800-645-6264 www.accurate-assi-leeches.com Amersham Pharmacia Biotech 800-323-9750 www.apbiotech.com AMRESCO 800-829-2805 www.amresco-inc.com Bio-Rad Laboratories 800-4-BIORAD www.bio-rad.com Biotech Holdings 216-655-5644 www.biotechholdings.com Diversified Biotech 800-796-9199 www.divbio.com ESA, Inc. 508-250-7000 www.esainc.com Geno Technology, Inc. 314-534-0075 genotech@genotech.com Goldmark Biologicals 908-859-2631 http://members.aol.com/goldmarker/ ICN Biomedicals 800-854-0530 www.icnpharm.com Life Technologies 800-828-6686 www.lifetech.com Molecular Probes 800-438-2209 www.probes.com National Diagnostics, Inc. 800-526-3867 www.nationaldiagnostics.com Novex 800-456-6839 www.novex.com Owl Separation Systems 800-242-5560 www.owlsci.com Pierce Chemical Company 800-874-3723 www.piercenet.com Roche Molecular Biochemicals 800-262-1640 http://biochem.boehringer-mannheim.com Sialomed, Inc. (now Amika Corp.) 800-547-6766 sialo@sialomed.com Sigma Chemical Company 800-325-3010 www.sigma.sial.com Zaxis 800-GEL-CODE www.zaxis-inc.com Sigma Chemical Company Brilliant Blue G-Colloidal Concentrate <10 ng/band $27.35 A colloidal suspension of Coomassie Blue G-250, this product has a tenfold increase in sensitivity after fixation of the proteins compared with the conventional methanol/acetic acid preparations of Coomassie Blue R-250 and G-250. The concentrate makes 1 liter of working solution, and is suitable for IEF (after fixing), PAGE and SDS-PAGE. Zaxis Colloidal Coomassie G-250 Stain 0.349 ng/mm2 $30.00 The Colloidal Coomassie G-250 Stain from Zaxis is as sensitive as the Merril Silver Stain procedure (described later) and is highly quantitative with minimal background. The use of image burst solution can further intensify protein bands. Destaining is optional. Recommended for most animal protein applications, this product will stain ten gels, but is reusable four to five times. Driven by the need to detect proteins in low or trace amounts, protein researchers began the search for more sensitive gel stains in the 1970's. In 1979, C.R.Merril (C.R. Merril et al., Proceedings of the National Academy of Sciences, U.S.A., 76:4335, 1979) reported the application of a histochemically derived silver stain to polyacrylamide gel electrophoresis that had a hundredfold increase in sensitivity over conventional methanol/acetic acid Coomassie Blue staining. Because of some drawbacks to this technique, however, Merril (C.R. Merril et al., Science, 211:1437-8, 1981) developed a photochemical silver stain technique in 1981 that has become the standard to which all other silver staining techniques are compared. This method and its variations have numerous features, including specificity for all types of macromolecules, application to various electrophoretic techniques, such as IEF, native, 2-D, gradient, and denaturing nucleic acid gels, and the ability to stain macromolecules in different colors ( The Bio-Rad Silver Stain. Bio-Rad Laboratories. Bulletin # 1089). Several modified silver staining techniques have been developed since the photochemical method of Merril was first described. These modified techniques offer simplified procedures, improved reagent stability, reduced background staining, lower costs, and other improvements when compared to the Merril method. A brief overview of the commercial silver staining kits based on these modifications follows. Accurate Chemical & Scientific Corporation Accurate Silver Stain Kit nanogram amts. $98.00 Based on the method described in Wray et al. ( Anal. Biochem., 118:197-203, 1981), the Accurate Silver Stain Kit uses a 45 minute staining procedure that permits user-controlled staining intensity. The kit contains enough reagents for 25 gels and may be used before or after Coomassie blue staining. The kit stains gels up to 3 mm thick, stains RNA and DNA in acrylamide gels, and is effective with the common PAGE methods. Amersham Pharmacia Biotech PlusOne Silver Staining Kit (Protein) 0.2-0.6 ng/band $90.00 PhastGel Silver Kit (Protein) 1-5 ng/band $119.00 The Silver Staining Kit, Protein provides sensitive and reproducible staining of proteins in PAGE gels in under two hours. Based on the method described by Heukeshoven and Dernick ( Electrophoresis , 6 :103-12, 1985), this kit produces nearly colorless backgrounds with most electrophoretic systems and will stain 20 mini gels. Typical sensitivities obtained are between 0.2 ng and 0.6 ng per band, but under certain conditions, greater sensitivities can be achieved and faster development times, down to 15 minutes, are possible. Sold specifically for Amersham Pharmacia's PhastSystem, the PhastGel Silver Kit is optimized for rapid and sensitive staining of proteins in PhastGel media using the PhastSystem Development Unit. This procedure detects 1 ng to 5 ng of native protein per band, and as low as 0.3 ng for SDS denatured proteins. Bio-Rad Laboratories Bio-Rad Silver Stain Kit 1 ng-10 ng $105.00 Silver Stain Plus Kit 1 ng-10 ng $128.00 Arguably the leader in silver staining, Bio-Rad's Silver Stain Kit is based on the photochemical silver staining method of Merril. This kit has wide-ranging and well-documented applications as discussed previously. Bio-Rad's Silver Stain Plus Kit is the most sensitive and is easy to use. Derived from the method of Gottleib and Chavko ( Anal. Biochem., 165:33-7, 1987), this kit features a modified chemistry that detects proteins and nucleic acids in both acrylamide and agarose gels. ESA, Inc. Minigel Silver Stain Kit to 2 ng $75.00 Investigator Silver Stain Kit to 2 ng $120.00 The Investigator and MiniGel Silver Stain Kits from ESA both use a modification of the Rabilloud procedure (T. Rabilloud, Electrophoresis , 13 :429-39, 1992). This procedure exhibits excellent sensitivity and a high signal- to-noise ratio, producing crisp bands or spots against a water-clear background. The use of silver nitrate as the silvering agent enables the quantitation of bands by densitometry when the development times are kept below 10 minutes. Less abundant bands may be detected by the extension of development times to 30 minutes without an appreciable gain in background. Geno Technology, Inc. FASTSilver 1 ng/band $125.00 The FASTSilver kit features a 60 minute to 90 minute protocol that produces sharp images of protein and nucleic acid bands against a clear background. The kit includes ready-to-use reagents and stains 25 mini gels. ICN Biomedicals Rapid-Ag-Stain 64 pg/band $335.45 Silver Stain Quick Kit 64 pg/band $42.00 ICN's Rapid-Ag-Stain is an improved silver stain reagent that stains gels in less than an hour. Suitable for proteins, RNA, DNA, and 2-D electrophoresis, the Rapid- Ag-Stain kit is 125 times more sensitive than Coomassie Blue and employs a simple three-step procedure. The kit contains reagents for staining 25 slab gels. The Silver Stain Quick Kit is a rapid and sensitive silver staining kit consisting of 100 ml of 10X silver stain oxidizer, 100 ml of 10X Silver Stain reagent, and 125 ml of Silver Stain Developer. Zinc Stain from Bio-Rad Gold-Blot from Owl Separation Systems SYPRO Orange from Molecular Probes SYPRO Red from Molecular Probes National Diagnostics, Inc. STERLING Rapid Silver Stain 5 ng protein $75.00 The STERLING Rapid Silver Stain kit from National Diagnostics uses a simple, three-step staining process to stain polyacrylamide gels in less than an hour. Suitable for both protein and DNA staining, bands are revealed by a 5 minute to 10 minute treatment in the silver reagent after fixing and washing steps. The STERLING kit stains 18 mini gels. Novex SilverXpress Silver Staining Kit 0.86 ng/band $126.00 The SilverXpress Silver Staining Kit uses ammoniacal silver chemistry to detect subnanogram amounts of protein in 90 minutes. The kit contains reagents for 25 mini gels and stains most electrophoretic systems. Owl Separation Systems Owl Silver Stain 0.1 ng/band $150.00 Owl Silver Stain from Owl Separation Systems is an improved ammoniacal silver stain kit that offers more control over the end point of image development. The kit produces a more monochromatic silver stain for enhanced sensitivity and easier scanning of 2D or any other type of polyacrylamide gel. The kit can stain 20 to 40 minigels. Pierce Chemical Company GELCODE Color Silver Stain Kit 0.1 ng/mm2 $142.00 Pierce Chemical Company's GELCODE Color Silver Stain Kit is a complete system for staining one- and two-dimensional SDS PAGE gels. Derived from the method described in Sammons et al. ( Electrophoresis, 2:135-41, 1981), the GELCODE Color Silver Stain Kit stains proteins, DNA, lipids, and polysaccharides in five basic colors. Sigma Chemical Company Silver Stain Kits $34.60/$102.90 Rapid Silver Staining Kit $102.20 Double Staining Kit $108.60 Sigma's Silver Stain Kits, consisting of a 5 gel capacity starter kit and a larger, 25 gel capacity kit, are based on the method of Heukeshoven and Dernick. Each has applications for gels between 0.5 mm and 3.0 mm thick, including SDS, urea, non-denaturing, and IEF gels. Both have low background staining properties and contain stable solutions. Staining protocols require about 3.5 hours and are simple and reliable. The Rapid Silver Staining Kit stains proteins and nucleic acids in polyacrylamide gels in 1.5 hours. This product contains sufficient reagents for staining 25 mini gels. Sigma's Double Staining Kit is the only kit in the profile to take advantage of enhanced silver staining following Coomassie Blue staining by combining a colloidal Coomassie stain and a silver stain into one product. The sensitivity of this kit is 10 times the level of silver staining alone. Zaxis, Inc. Zaxis Silver Stain 0.1 ng/mm2 $30.00 The Zaxis Silver Stain from precast gel manufacturer Zaxis, Inc. detects protein, DNA, and RNA with a detection limit of less than 0.1 nanogram per square millimeter of gel. This detection limit drops even further when the Zaxis Silver Stain is used following Coomassie Blue staining. Each kit stains 15 PAGE or agarose gels following a conventional silver stain method in under two hours. While considerable effort has been spent developing sensitive stains for detecting proteins in gels, the most popular ones, Coomassie and silver, do not lend themselves to efficient recovery of proteins from the gel matrix. In response to this problem, C. Lee et al. ( Anal. Biochem., 166:308-12, 1987) reported a copper chloride stain that reversibly fixes proteins in SDS PAGE gels. Because the stain is rapidly removed by chelation with EDTA, the proteins can be quantitatively eluted or electrotransferred after destaining. Staining occurs when copper ions react with the Tris and SDS in the gel matrix to produce a negatively stained gel consisting of clear protein bands against a semiopaque whitish-blue background. Staining is complete in 5 minutes. The protein bands are visualized by placing the gel against a dark background and are easily photographed. Copper staining also provides a moderate increase in sensitivity compared with Coomassie staining. The method described in Lee et al. has given rise to three rapid and reversible gel staining products. All three produce stained SDS PAGE gels in 5 minutes. Bio-Rad's Copper Stain and Destain Kit ($49.00) includes both stain and destaining reagents and makes 1.25 liters of stain solution. The 5 Minute Reversible Stain™ from Geno Technology, Inc. ($95.00) includes enough stain and destainer for 25 mini gels. The destaining solution is sold separately. Finally, Sigma's Rapid Reversible Stain ($88.05) makes one liter of staining reagent and is sold separately from the destaining reagent. The use of copper and other heavy metal gel stains was explored further by J.K. Dzandu et al. (Anal. Biochem., 174:157-67, 1988), who reported the use of zinc chloride as another negative, reversible staining agent. C. Fernandez-Patron et al. ( BioTechniques, 12:564-73, 1992) improved zinc chloride staining with the incorporation of an imidazole treatment step before the application of the stain. Here, bands are developed in 30 seconds to 60 seconds after a 10-minute imidazole treatment. Like copper staining, zinc stained gels are readily destained allowing blotting, amino acid analysis, and sequencing to be performed on the samples. Bio-Rad has applied the techniques of Fernandez-Patron et al. to its Zinc Stain and Destain Kit ($80.00). This kit includes both stain and destain, and has sufficient reagents to stain 50 mini gels. The final entry into the reversible staining kit category is the Reversible Protein Detection Kit from Sigma Chemical Company ($53.00). After thorough fixing and washing steps, gels are stained in a dark purple reagent for 20 minutes to 40 minutes; the entire process requires about 3.5 hours for SDS PAGE gels and about 2.5 hours for native gels. Destaining is performed by incubation in a 50 mM EDTA solution. Molecular Probes SYPRO® Red Stain 1-2 ng on film 50 ng by eye; $100.00 SYPRO® Orange Stain 1-2 ng on film 50 ng by eye; $100.00 Bio-Rad Laboratories SYPRO® Orange Stain 1-2 ng $99.00 Molecular Probes has tackled protein detection in gels with the use of two fluorescent dyes called SYPRO Red Stain and SYPRO Orange Stain. Rivaling silver staining in sensitivity, these two dyes are much easier to use and have applications with SDS PAGE (T.H. Steinberg et al., Anal. Biochem., 239:223-97, 1996), two-dimensional gels, native gels, and as non-interfering stains for Western blotting and the immunodetection of blotted proteins (T.H. Steinberg et al., Anal. Biochem., 239:238-45, 1996). At the completion of electrophoresis, gels are stained by a 30 minute to 60 minute incubation in a 1:5000 dilution of either dye in 7.5 percent acetic acid. After a brief 7.5 percent acetic acid rinse, the proteins are visualized by transillumination with 300 nm UV light on a UV light box. The fluorescent signals are recorded by Polaroid Type 667 film, CCD cameras, or commercially available laser scanners. Because the SYPRO stains bind to the SDS coats on proteins, and not to the proteins themselves, the stains show very little protein-to-protein signal variation. The SYPRO stains also demonstrate a broad linear response to protein amounts, stretching across three orders of magnitude of protein per band. This broad response has important applications to the quantitation of proteins in gels. SYPRO dyes appear to bind noncovalently to protein-SDS complexes and thus do not mask antigenic sites. By switching to nonfixative staining procedures, such as staining gels in transfer buffer, Molecular Probes has shown that gels may be stained with the SYPRO dyes prior to electrotransfer without hindering transfer or subsequent antibody detection (T.H. Steinberg et al., Anal. Biochem., 239:238-45, 1996). These dyes therefore provide researchers with the ability to stain and record gels prior to immunoblotting experiments. The SYPRO dyes cost $100 for 0.5 ml dye, which provides stain to prepare 2.5 liters of working solution, sufficient to stain 50 gels if the solution is used once. SYPRO stain can be reused up to four times, however, which means that 50 to 200 gels can be stained with the supplied stock unit of dye. SYPRO ORANGE stain is also available from Bio-Rad Laboratories. ICN Biomedicals Fast Green FCF 25g/$28.40 Stains-All 5 g/$113.30 Uniblue A 1 g/$27.15 Fast Green FCF is an acid dye with important application for densitometry. Fast Green stains proteins with a wide linear relationship between protein concentration and color density, unlike Coomassie Blue which deviates from Beer's law at higher protein concentrations (M.A. Gorovsky et al., Anal. Biochem., 35:359-70, 1970). A cationic carbocyanine dye was found to exhibit changes in its absorption spectrum when bound to various macromolecules. For example, this dye stains proteins red, RNA bluish purple, and DNA blue. This ability earned the dye the name "Stains-All." Stains-All has been used to distinguish phosphorylated and non-phosphorylated proteins on the basis of their staining colors (M.R. Green et al., Anal. Biochem., 56:43-51, 1973). Uniblue A, a derivative of the acid wool dye Remazol Brilliant Blue R, also stains proteins with a broad linear dye to protein relationship (A. Datyner, E. Finnimore, Anal. Biochem., 52:45-55, 1973). The visualization of proteins blotted onto transfer membranes has important applications for the localization of bands wanted for sequencing and the comparison of individual proteins detected by immunostaining with the total population of proteins found on duplicate companion blots. Staining methods include colloidal gold, reversible stains, and Coomassie Blue. Diversified Biotech Membragold 1 ng/band $120.00 Goldmark Biologicals PROTOGOLD 1 pg $120.00 Bio-Rad Laboratories Colloidal Gold Protein Stain 1 ng/band $79.00 Gold Enhancement Kit 10 pg $140.00 Owl Separation Systems Gold-Blot(TM) 1 ng/band $120.00 Consisting of colloidal gold staining solutions, these products stain proteins bright red through the accumulation of gold particles bound to the proteins by hydrophobic and ionic interactions. Because of the negatively charged nature of colloidal gold, these stains are suitable for use only with negatively charged membranes such as nitrocellulose and PVDF. Positively charged nylon membranes bind colloidal gold nonspecifically, producing high backgrounds. Goldmark Biologicals' PROTOGOLD reveals bands in 10 minutes with optimal staining occurring after 2 hours. The sensitivity of PROTOGOLD is as low as 1 pg of protein and overstaining does not occur. Goldmark also sells the BBI BL Silver Enhancing Kit which enhances the signal intensities of gold-stained blots 10 times to 100 times. Diversified Biotech's Membragold solution consists of a unique formulation based on gold colloid concentration and particle size distribution considerations. The result is a high sensitivity stain that produces optimal staining in 30 minutes to 60 minutes with no increase in background intensity. Bio-Rad's Colloidal Gold Total Protein Stain is billed as a very rapid and sensitive stain, detecting 1 ng of protein on PVDF and nitrocellulose membranes. The optional silver-based Gold Enhancement Kit increases sensitivity to as little as 10 pg. Gold-Blot(TM) from Owl Separation Systems is a colloidal gold reagent for total protein staining on nitrocellulose or PVDF membranes. Gold-Blot complexes with proteins to produce red-purple bands in thirty minutes. This kit can stain 10 membranes and includes a detergent blocking agent for minimal background and ready-to-use stain reagent. Geno Technology, Inc. BLOT-FastStain 0.5 ng/band $130.00 Sigma Chemical Company Reversible Protein Detection Kit $53.00 ESA, Inc. Rev-Pro Reversible Protein Stain Kit to 25 ng $75.00 The transfer membrane staining products offered by Geno Technology, Sigma, and ESA, are all billed as rapid and reversible. The BLOT-FastStain from Geno Technology reversibly stains proteins on nitrocellulose and PVDF membranes. The 10-minute staining procedure leaves dark bands against an untouched white background. Complete destaining is accomplished by a 10-minute rinse in warm water, leaving proteins available for immunoblotting and sequencing protocols. Sigma's Reversible Protein Detection Kit is the only membrane-staining product in the profile that stains nylon membranes in addition to nitrocellulose and PVDF. The staining procedure takes about 45 minutes and has sensitivity comparable to Coomassie Blue acrylamide gel staining. Destaining involves a 5- to 30-minute wash in an EDTA solution, and leaves the blot ready for immunostaining procedures. The Rev-Pro stain from ESA is a sensitive, fully reversible stain for proteins blotted onto nitrocellulose or PVDF membranes, and is compatible with immunostaining, protein sequencing, and mass spectrometry protocols. Rev-Pro stain does not covalently modify proteins, cause N-terminal blocking, interfere with the recognition site for trypsin or other proteases, nor mask the antigenic recognition sites for antibodies. Both Pierce Chemical Company's GelCode Blue Stain Reagent and Diversifed Biotech's Rapid Coomassie Stain (described previously) have transfer membrane staining applications. GelCode Blue stains PVDF membranes in under an hour with a follow-up water enhancement step. The Rapid Coomassie Stain stains proteins on nitrocellulose and PVDF in 5 minutes to 10 minutes. Amika Corp. Cozap destaining sheet 25 for $59.00 Diversified Biotech Stain-Out destaining sheet 25 for $55.00 ICN Biomedicals Destaining Sponge destaining sponge 50 for $25.15 Novex Gel-Clear Destaining Solution destaining solution 250 ml for $51.45 Four companies sell products designed to accelerate and simplify gel destaining procedures for gels stained with Coomassie Blue. Three products, Cozap, Stain-Out, and the Destaining Sponge are all used in a similar way. When added to gel destaining solutions, each binds up free Coomassie Blue, extending the lives of those solutions many times by eliminating the need for repeated solution changes. Cozap, a white, foam-like pad material, has a high affinity for Coomassie Blue and leaves gel backgrounds and destain solutions crystal clear. Each pad is sufficient for destaining 10 mini gels. Stain-Out is an activated carbon filter sheet that not only absorbs Coomassie Blue, but removes Amido Black, Ponceau S, ethidium bromide, histochemical stains, and radioactive compounds from solution as well. A 2-inch x 3-inch piece of Stain-Out will bind up to 20 milligrams of Coomassie Blue. ICN's Destaining Sponge has a high affinity for Coomassie Blue and efficiently and quickly attracts it, reducing destaining time and methanol needs. One 5 cm x 5 cm sponge is used per gel. Novex has taken a different path with its Gel-Clear Destaining Solution. Designed specifically for Coomassie R-250/acetic acid stains, Gel-Clear Destain traps stain molecules in solution, enabling the visualization of bands within 15 minutes and eliminating the need for solution changes. One 250 ml bottle is plenty for destaining at least 30 gels. Amersham Pharmacia Biotech Hoefer Automated Gel Stainer $3,950.00 Automation hits general purpose gel staining with the Hoefer Automated Gel Stainer from Amersham Pharmacia Biotech. Designed for the automated multistep staining of unbacked and plastic-backed polyacrylamide gels, this instrument features efficient reagent usage and the ability to segregate hazardous and nonhazardous wastes using its 10-port valve. Preprogrammed with three silver and four Coomassie staining programs for protein gels, and one silver staining program for DNA gels, the Automated Gel Stainer also stores up to 10 user-defined programs. Each program runs up to 20 steps and accommodates nine reagents. The Hoefer Automated Gel Stainer is available with a choice of staining tray sizes to accommodate various gel size formats. Staining times run from a low of 100 minutes for silver staining 1.0 mm unbacked DNA gels, to a high of 460 minutes for Coomassie staining of 1.5 mm thick protein gels. A rich history of staining technique development has accompanied the growth of polyacrylamide gel electrophoresis and has contributed greatly to the protein staining products available to the life scientist. As staining methods become faster, easier, and more sensitive than ever before, we hope the information provided here will aid you in your protein research efforts. Michael Brush is an Associate Scientist and Radiation Safety Officer at Hitachi Chemical Co. in Irvine, Calif. He can be reached online at MDBrush@compuserve.com.

Dye Hard: Protein Gel Staining Products

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