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From: Cell Biology: A Laboratory Handbook. 3rd Edition. v.4 Eds: Celis, J.E., Carter N., Hunter T., Simons K., Small J.V.

and Shotton D. 2006. Elsevier. Academic Press.

Protein Detection in Gels by Silver Staining: A Procedure Compatible with Mass-Spectrometry
I. Gromova and J.E. Celis
I. INTRODUCTION

Silver staining is one of the procedures, in addition to Coomassie Blue, R and G types, (Neuhoff at al., 1988) and fluorescent dyes (Steinberg at al., 1996; Patton, 2002; see also article by Patton in this volume) that are available for detecting proteins separated by gel electrophoresis. Switzer et al., introduced silver staining in 1979, a technique that today provides a very sensitive tool for protein visualization with a detection level down to the 0.3-10 ng level (Switzer at al, 1979). The basic mechanisms underlying silver staining of proteins in gels are relatively well understood. Basically, protein detection depends on the binding of silver ions to the amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins (Switzer at al, 1979; Oakley at al., 1980; Merril at al., 1981; Merril at al., 1986), followed by reduction to free metallic silver (Rabilloud, 1990; Rabilloud, 1999). The protein bands are visualized as spots where the reduction occurs and, as a result, the image of protein distribution within the gel is based on the difference in oxidation-reduction potential between the gel’s area occupied by proteins and the free adjacent sites. A number of alteration in the silver staining procedure can shift the oxidationreduction equilibrium in a way that gel-separated proteins will be visualized either as positively or negatively stained bands (Merril at al., 1986). Silver staining protocols can be divided into two general categories: 1) silver amine or alkaline methods, and 2) silver nitrate or acidic methods (Merril, 1990). In general, the detection levels’ using the various procedures is determined by how quickly the protein bands develop in relationship to the background (e.g. signal-to-noise ratio). The silver amine or alkaline methods usually have lower background and as a result are most sensitive but require longer procedures. Acidic protocols, on the other hand, are faster but slightly less sensitive. A comparative analysis of the sensitivity of a number of silver staining procedures has been published recently (Sorensen et al., 2002; Mortz et al., 2001). Clearly, each protocol has different advantages regarding timing, sensitivity, cost, and compatibility with other analytical methods, especially mass-spectrometry (MS) a tool that is

III. Use high quality laboratory reagents that can be purchased from any commonly used chemical company. Until recently most of the silver staining protocols used either glutaraldehyde. and subjected to MS analysis (Shevchenko et al. To make 1 liter.. add 120 ml of glacial acetic acid to 500 ml of 96% ethanol and 500 µl of 35% formaldehyde (note: commercial formalin is 35% formaldehyde). recovered from the gel. thus. mix well and bring to the final volume of 1 liter .05% formalin. 2. Washing: 20% ethanol (or methanol). Sensitizing solution: 0. Several modifications of the silver nitrate staining procedure have been developed for visualizing proteins that can be subsequently digested. II. add 200 ml 96% ethanol to 800 ml of deionized water.02% (w/v) sodium thiosulfate (Na2S2O3). Store at room temperature. Complete to final volume with deionized water. that is high sensitivity and low background. 2000). Store at room temperature. Solutions 1. 1990). 3. it is very important to follow closely the incubation time of all steps as given in the protocol. introducing the chemical modifications into proteins. 0.of formaldehyde-based sensitizes in the fixing and sensitization step.2 being used in combination with gel electrophoresis or chromatographic methods for rapid protein identification (see various articles in this volume). 1996. PROCEDURE To achieve the best results. Fixation solution: 50% ethanol (or methanol). 12% acetic acid. To make 1 liter. Yan at al. To make 1 liter add 200 mg of sodium thiosulfate anhydrate to small volume of deionized water. In this article we describe a procedure that is slightly modified from these. strongly affecting two most crucial steps for the subsequent MS analysis: hampering the trypsin digestion and highly reduces the protein extraction from the gel (Rabilloud.. thus. MATERIALS AND INSTRUMENTATION Ultra pure water (> 18 megom/cm resistance) for preparation of all buffers as well as during the washing steps is recommended. The utilization of those chemicals causes the cross-linking of two lysine residues within protein chain.

Dissolve and bring to final volume with deionized water. 0. Add 4 mg of sodium thiosulfate anhydrate to a small volume of deionized water and dissolve. To make 1 liter. For each step use sufficient volumes of the solutions to fully immerse the gels. Terminating solution: 12% acetic acid.05% formalin. Pre-cool the solution at 4oC before using. The gel fixation and washing procedure can be carried in a staining try (polyproylene trays are recommended) but make sure that these are only used for silver staining. Has to be prepared fresh. remove the gel from the cassette and place into a tray containing appropriate volume of fixing solution.2% (w/v) silver nitrate (AgNO3).076% formalin. Staining: 0. Soak the gel in this solution app. Wash the gloves with water during the staining procedure.0004% (w/v) sodium thiosulfate (Na2S2O3). 5. Store at room temperature. 2 h. The staining procedure can be performed on any type of the rotary shaker. add 120 ml of glacial acetic acid to 500 ml of deionized water. 0. placed on a shaker at a very gentle speed. Steps Use powder free rubber gloves throughout the procedure. Fixation will restrict protein movement from the gel matrix and will remove interfering ions and detergent from the . 0. one gel per tray. Do not touch the gel with the bare hands or metal objects during the handling. Store at room temperature. After electrophoresis. To make 1 liter add 2 g of AgNO3 to a small amount of deionized water. Drying solution: 20% ethanol. Mix well. Add 760 µl of 35% formaldehyde. 1. Close the plastic trays by the lead or place the trays on the top of each other to protect the gels from dust. The size of the container has to be big enough to perform the free movement of the gel during the shaking. 6. To make 1 liter.3 4. Developing solution: 6% (w/v) sodium carbonate (Na2CO3). add 200 ml of ethanol to 800 ml of deionized water. Mix both solutions. Plastic or Teflon bars (or ordinary glass pipettes) can be used to handle the gel. 7. add 500 µl of 35% formaldehyde and bring to the final volume with the water. Mix well and bring to the final volume with water. Perform all steps at room temperature. Store at room temperature. To make 1 liter add 60 g Na2CO3 to a small amount of deionized water and dissolve.

. Discard the ethanol solution and add enough volume of the sensitizing solution. 3. Discard the solution. If water is used during the washing step the size of the gel can be restoring by incubation of the gel in 20% ethanol for 20 min. 5. Discard the fixative solution and wash the gel in 20% ethanol for 20 min. Note: Washing the gel for more than one min will remove the silver ions from the gel resulting in decreased sensitivity. Stop the reduction reaction by adding 50 ml of terminating solution directly to the gel that is still immersed into developing solution. 10. pour off the staining solution and rinse the gel with a large volume of deionized water to 20-60 sec to remove the excess of unbound silver ions. 7. 1 min each time.4 gel. As soon as “boubling” of the solution is over. Add the solution to the corner of the tray. 9. Change the solution three times to remove the remaining detergent ions as well as fixation acid from the gel. Repeat the washing once more. in this volume. 8. Fixation can also be done overnight. Since the sensitivity of silver staining is in the same range as the modern mass . The reaction can be stopped as soon as the desired intensity of the bands is reached. Incubate for 2 min with gentle rotation. B shows silver stained gels of total protein extracts from normal colon tissue biopsy and a breast tumor biopsy separated by 2D gel electrophoresis as described by Celis et al.5 min. the development is stopped. 2. After staining is complete. Discard the water. Moist gels can be kept in 12% acetic acid at 4oC in sealed plastic bags or placed in the drying solution for 2 hr prior to vacuum drying. It may improve the sensitivity of the staining and decrease the background. Add new portion of the developing solution and develop the protein image by incubation the gel in 300 ml of developing solution for 2 . Add the cold silver staining solution and shake for 20 min to allow the silver ions to bind to proteins. Gently agitate the gel during 10 min. It will increase the sensitivity and the contrast of the staining. Figures 1A. Note: We recommend using ethanol solution instead of deionized water to prevent gel’s swallowing. 4. Rinse the gel shortly with the developing solution. Note: Do not pure the staining solution directly on the gel as it may result in unequal background. in deionized water. 6. Discard the sensitizing solution and wash the gel twice.

it makes this staining one of the most attractive technique for protein visualization before the MS analysis. B.5 spectrometry. . Protein bands of various intensity can be excised from the gels and identify by MS analysis (see various articles in this volume). A.

The . location 7. can be increased by distaining of the silver stained protein bands (Gharahdaghi at al. 1. 10-fold range. V. UK and SilverQuestTM Silver Staining Kit. 2. and some of very acidic proteins are detected poorly by silver staining (Goldberg H. Thus. thus hampering the use of this method for quantitative protein expression analysis.A. Amersham. wash the gel longer after fixation to remove all residual acid. This extra washing will reduce the background during development.. Development of the gel for a long period of time can decrease the yield of the peptides for subsequent mass-spectrometric analysis. including proteoglucans and mucins. 4. 3. Recently it was published that the recovery of peptides from the gel. at a. measurement and database analysis.6 Figure 1 2D gel of normal human colon. MS analysis of proteins resolved by gel electrophoresis utilizes the extraction of protein spot from the stained gel followed by trypsin digestion. for MS analysis. USA. PITFALLS To increase the sensitivity of the staining. Note. which contain high levels of sulfated sugar residues. Several silver staining kits that offer improved compatibility with subsequent mass spectrometric analysis are commercially available. these include: Silver Stain PlusOne. IV. that linear dynamic range of the stain is restricted to app. artificial bands with a molecular mass of around 50-70 kDa as well as streaking or yellow background can be observed due to the presence of high concentration of reducing agents such as 2-mercaptoethanol or DTT in the sample buffer. Negative staining can be observed when an excess of protein is applied. 1997). several classes of highly negative charged proteins. 5. This is due to the fact that mainly unstained peptides from the inner part of the gel are eluted to the solutions following “in gel” tryptic digestion” of proteins (see article in the volume). 1999).l. Protein spots labeled on the gel images were identified by MALDI-TOF-MS analysis. COMMENTS When choosing the silver staining protocol it is necessary to remember that not all proteins are equally staining by this technique. Amersham Pharmacia Biotech. In some cases. (A) and human breast tumor biopsy (B) separated by 2D-gel IEF electrophoresis and stained with silver nitrate. Invitrogen.

V.. (1999).A. Ehrhardt. A. Pratt. 251(2)..7 unpublished observation is indicated that it will only be the case if the distaining is performed immediately after the staining procedure. D.. Anal Biochem. Warner. S. D.. C.. 601-5.R. 96-110. 255-62. 343(6260). M. Oakley. Anal Biochem. Dunau.S.R. Vorum. A rapid sensitive silver stain for polypeptides in polyacrylamide gels. D. C.R. C.. Taube. Nature. 105(2). Gorg. Imai. Goldman.. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.. K. (1981). N. Krogh. C. H. E.R.. Merril. 110(1). D. (2001). Anal Biochem. Neuhoff. Mische. F.J.E. . Arold. 20(3). M. Merril. (1988I). (1986)...L. Morris. Merril. 156(1). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. (1980). 227-33.N. 779-80. 9(6). Weinberg. W. Kirsch.. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Electrophoresis. T. 201-7. The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate. Anal Biochem. A silver stain for the rapid quantitative detection of proteins or nucleic acids on membranes or thin layer plates. (1997).A.. Mortz. Meagher.R. 1359-63. 361-3. B.... Goldberg. Electrophoresis.R. B.R. 1(11).M. H. (1990). N. Proteomics. Silver staining of proteins and DNA.. References Gharahdaghi.

R. T. 3341. SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein.. C. Enghild. Jones. J.8 Patton. 771(1-2)... M. Steinberg..H. Westbrook. Switzer. Singer. 112.K.F. Anal Biochem. Merril. Ostergard. 3-31. 17.S..X. 231-7..A. Wait.J.P.. . Anal Chem. Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis. M. A. 3rd.. Shevchenko. (999). P. (2002). Sorensen. Detection technologies in proteome analysis... R. Wheeler. Electrophoresis. Rabilloud. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. L. 10.C. O. G. Haugland. Hojrup. Wilm.R. R. M. Shifrin. Ryder. R. Mann... Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis. (1979). T. C. Harry. V. Dunn. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Houen. Rabilloud.. Vorm. Anal Biochem. Anal Biochem. 304(1). Methods Mol Biol. (1990). L.H. E.. Electrophoresis. 239(2).J..R. 98(1). Berkelman. 3666-72.. T. 223-37. C. Yan. B. T.A. Jorgensen.. 785-94. (1996).... W. A modified silver staining protocol for visualization of proteins compatible with matrixassisted laser desorption/ionization and electrospray ionization-mass spectrometry. (1996). S. J. 297-305. 68(5). J Chromatogr B Analyt Technol Biomed Life Sci. (2000). Silver staining of 2-D electrophoresis gels. J.L. (2002). 850-8.