IMPROVED SILVER STAINING METHOD FOR DETECTING BIOMOLECULES AND KIT THEREFOR

Abstract: The invention relates to a method of staining biomolecules present in or on a support medium, which method comprises fixing the biomolecules to the support medium, treating the support medium with a silver solution, and developing the thus-treated support medium with a reducing agent. Fixing of the biomolecules and treatment with silver solution are performed in a single step by impregnating the support medium with a silver solution containing silver ions and a strong acid which has a pK value lower than 3 and which at the impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution. The invention also relates to a kit for carrying out the method.

Description: IMPROVED SILVER STAINING METHOD FOR DETECTING B OMOLECUI-ES AND KIT THEREFOR FIELD OF THE INVENTION The present invention relates to an improved silver staining method for detecting biomolecules like proteins, peptides, nucleic acids and saccharides after separation on a support medium by gel electrophoresis or other techniques. The invention also relates to a kit for carrying out the method. BACKGROUND OF THE INVENTION The analysis of proteins and other biomolecules often include separation by electrophoretic techniques on a support medium, such as polyacrylamide gel. The electrophoresis results in a migration pattern which may be defined by various methods, including visual detection methods. The visualisation methods include staining procedures, the most commonly used procedures being Coomassie blue staining, ethidium bromide staining and silver staining. Silver staining was originally introduced as a detection method for proteins in polyacrylamide gels (Switzer, R.C., Merrill, C.R., and Shifrin, S. (1979) Anal . Biochem 9Ji, 231-237) and comprises treating the gel with a silver salt solution and then developing the treated gel with a reducing agent to transform the silver ions into collodial silver grains. The use of the method has been extended to include also the detection of nucleic acids and peptides. Gel media used today include agarose and gels based on different types of acrylamide derivatives. The great advantage of silver staining compared to alternative staining methods using, for example, Coomassie Brilliant Blue or ethidium bromide is the very high sensitivity obtained. Since the introduction of the method several hundred articles dealing with silver staining have appeared and a large number of protocols describing different silver staining methods have been published. These protocols are normally divided into two groups based on whether silver nitrate or silver diamine is used for silver impregnation (Rabilloud, T. (1992) Electrophoresis H, 429-439) . When silver nitrate is used for impregnation, the reduction is done with formaldehyde at basic pH values, normally established with sodium or potassium carbonate. In many of the more recent protocols, sodium thiosulfate is included in the solution used for the development. With silver diamine impregnation, reduction is normally done with a solution containing formaldehyde and a small amount (~0.1 g/litre) of citric acid. In all silver staining protocols, silver impregnation is preceded by a number of steps designed to fix the biomolecules, remove buffer components from the gel and increase the sensitivity of the staining method. Numerous variations exist between published protocols. Fixation with a trichloroacetic acid solution seems to be preferable when isoelectric focusing or native protein electrophoresis is the separation method used (Heukeshoven, J., and Dernick, R. (1985) Electrophoresis £., 103-112), while alcohol-acetic acid solutions seem to be preferred with SDS-electrophoresis and for nucleic acid separation. Ethanol or propanol may just as well be used as methanol in the fixation solution. The fixation is followed by a pre-treatment step intended to improve the sensitivity of the stain. Normally, reducing compounds like glutardialdehyde, dithiothreitol or thiosulphate are used as sensitisers, but oxidative conditions (chromate-nitric acid) have also been utilised in the pre-treatment step. Common to almost all protocols is the fact that with intermediate washing steps included, a very large number of solution changes are required. This makes silver staining a time consuming and work intense method. The protocol according to Merril, C.R., Goldman, D. and Van Keuren, M.L. (1982) Electrophoresis 1, 17-23 given in Table 1 below (at the end of the description) , has been described as a fast and simple method, but still takes at least 1.5 hours and requires at least 10 changes of solution. Bassam, B.J., Caetano-Anolles, G., and Gresshoff, P.M. (1991) Anal . Biochem . 196. 80-83, have in connection with staining of nucleic acids eliminated the pre-treatment step and cut the total time for the protocol to 60-70 minutes (see Table 1) and still managed to maintain good sensitivity. Biel, H.J., Gronski, P., and Seiler, F.R. (1986) Electrophoresis 1_, 232-235 have used high temperature (60- 70°C) for fixation and combined fixation with silver impregnation in order to drastically reduce the time needed for silver staining. The solution used contained either only 2-3 g AgNC>3/litre or 2-3 g AgNC^/litre and 1.5 % acetic acid. The fixation and silver impregnation step (5- 10 min) was followed by a short wash (10-30 seconds) in deionized water at room temperature. The gel was then developed at 60-70°C with formaldehyde in a 0.5 M solution in NaOH. The drawback of this approach, besides the need to use high temperatures, is the low sensitivity obtained. Two preceding fixation steps (5 minutes in 1.5 % acetic, 50 % ethanol followed by 5 minutes in 1.5 % acetic acid, 80-90 % ethanol) , also at 60-70°C, improve the sensitivity, but according to the authors, there is only a thin layer at the gel surface that is silver stained. The time required for the preceding fixation steps need to be increased to 10 minutes each if the gel contains ions which precipitate silver. With this precaution the staining time falls in the range of 30-40 minutes in spite of the high temperature used. SUMMARY OF THE INVENTION It is an object of the present invention to simplify silver staining by minimising the number of steps and the time needed, while maintaining or improving the high sensitivity typical of most silver staining methods earlier described. To achieve the above-mentioned object, the two most time consuming steps in silver staining, viz. fixation and silver impregnation, have been combined into one step. The solution used in the combined fixation-impregnation step is titrated down to an acidic pH with a strong acid in order to maintain a high sensitivity. The demand put on the acid used is that the anion corresponding to the acid should neither form strong complexes with the silver ions nor precipitate silver under the conditions prevailing in the fixation-impregnation solution. In one aspect, the present invention provides a method of staining biomolecules present in or on a support medium, which method comprises the steps of fixing the biomolecules to the support medium, treating the support medium with a silver solution, and developing the thus- treated support medium with a reducing agent. The method is characterised in that fixing of the biomolecules and treatment with silver solution are performed in a single step by impregnating the support medium with a silver solution containing silver ions and a strong acid which has a pK value lower than 3 and which at the silver impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution. In another aspect, the present invention provides a silver staining kit comprising: (i) a fixing and silver solution comprising an acid having a pK value lower than 3 and which at the silver impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution, and a silver salt; (ii) a reducing agent comprising formaldehyde and, optionally, a thiosulphate; and (iii) a stop solution. Other objects and advantages of the invention are apparent from the following description. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a photograph of a separation gel silver stained by a prior art method; Figs. 2 to 4 are photographs of separation gels silver stained according to the invention using a silver solution containing perchloric acid, sulphosalicylic acid and benzenesulphonic acid, respectively; Fig. 5 is a photograph of a separation gel silver stained using a silver solution containing lactic acid; Figs. 6, 8 and 10 are photographs of separation gels silver stained according to the invention without pre-wash and using a silver solution containing perchloric, trichloroacetic and nitric acid, respectively; Figs. 7, 9 and 11 are photographs of separation gels corresponding to those of Figs. 6, 8 and 10, respectively, but silver impregnated after a preceding pre-wash; Figs. 12 and 13 are photographs of urea-containing separation gels silver stained according to the invention using a silver solution containing perchloric acid and naphthalenedisulphonic acid, respectively; Fig. 14 is a photograph of a urea-free separation gel silver stained according to the invention using a silver solution containing perchloric acid; and Fig. 15 is a photograph of a urea-free separation gel silver stained according to the invention using a silver solution containing perchloric acid, and where a different buffer was used in the electrophoresis than that used for the electrophoresis of the gels shown in Figs. 1 to 14. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, the methods currently used for silver staining of biomolecules, like proteins, peptides and nucleic acids, are simplified and speeded up by combining the fixation of the biomolecules with the silver impregnation into a single step. This is achieved by insuring fixation of the biomolecules by including a strong acid having a pK value lower 3 in the silver ion solution used for the impregnation. Any sufficiently strong acid may be used provided that the anion of the acid can neither form strong complexes with silver nor precipitate silver at the pH of the fixation- impregnation solution. Examples of suitable acids are nitric acid, hydrofluoric acid, chloric acid, perchloric acid, trichloroacetic acid, trifluoroacetic acid, methanesulphonic acid, benzenesulphonic acid, naphthtalenedisulphonic acid and sulphosalicylic acid. The pH, resulting after equilibration of the silver- containing solution with the gel to be stained, should be in the pH range of about 0.2 to about 3.0, and more preferably from about 0.7 to about 2.5. To achieve this, the concentration of strong acid in the fixation- impregnation solution should be in the range of about 0.004 to about 1 M, and the highest sensitivity will result with acid concentrations somewhere between about 0.01 and about 0.4 M. The minimum acid concentration giving good sensitivity will depend on the ratio of fixation- impregnation solution volume to gel volume and on the buffer composition of the gel. The low concentration limit given above is valid for high volume ratios (> 80) or when most of the gel buffer has been eliminated with a short pre-wash. With smaller volume ratios, and especially when the gel buffers contain high concentrations of weak bases, the lowest useful acid concentration is higher. As an example, gels run with Laemmli's buffer system ( aemmli, U. K. (1970) Nature (London) 277. 680) require a minimum concentration of about 0.04 in the fixation-impregnation solution for a solution to gel ratio of 10. The low pH value is essential for fixation of proteins and nucleic acids, but it also decreases the possibility for buffer substances present in the gel to form complexes with silver. The formation of stable silver ion complexes is connected to the ability of the silver ion to form electron pair bonds, and in the case of bases there is a direct competition between complex formation with silver and the addition of hydrogen ions to the base. The low pH will counteract formation of silver complexes with substances like tris, acetate, glycine, borate and tricine, which all may be present in the gel. The silver concentration of the fixation-impregnation solution may be in the range of about 0.4 to about 25 grams/litre, and generally it is preferable with a silver concentration between 1 and 5 grams/litre. Suitably, silver is added to the fixation-impregnation solution as silver nitrate, but in fact any silver compound capable of being solubilised in the fixation-impregnation solution to give the required silver concentration may be used. The fixation-impregnation solution may, in addition to silver ion and the strong acid, contain from about 10 to about 60 %, preferably from about 30 to about 50 % (by volume) alcohol. The presence of alcohol will improve the fixation of proteins and nucleic acids. The alcohol may, for example, be selected from methanol, ethanol, n-propanol and isopropanol. If desired, and if time is not critical, increased sensitivity may be obtained by performing a pre-fixing step with a solution of the strong acid, optionally containing alcohol as defined above, prior to the combined fixation- impregnation step. Agents capable of increasing the sensitivity of the silver staining, i.e. sensitisers, may also be included in the combined fixation-impregnation solution. Potassium or sodium dichromate addition can be combined with the use of nitric acid, while formaldehyde, glutardialdehyde or thiourea can be used in combination with a non-oxidising acid like hydrofluoric acid. The combined fixation-impregnation step according to the present invention may be directly followed by development or, optionally, a short (0.5 - 1 minute) intermediate wash in deionised water may be used. The development is done with a reducing agent capable of reducing silver at basic pH, preferably generated with sodium carbonate. Suitable sodium carbonate concentrations are in the range of about 20-30 grams/litre, and generally formaldehyde is an appropriate reducing agent. Useful formaldehyde concentrations are in the range of about 0.1- 1 grams/litre. The formaldehyde concentration is adjusted to give a convenient development time somewhere in the range of about 2-15 minutes. The development time will also depend on the silver concentration used in the fixation- impregnation step, and, generally, a high silver concentration corresponds to a low formaldehyde concentration in the development solution. Reduction of the silver concentration results in a need to increase the formaldehyde concentration for a suitable development time to be maintained. In order to avoid background staining of the gel, small amounts of thiosulphate may be added to the development solution in accordance with what has been described previously in the art for other silver staining methods. The development of the gel is stopped by commonly used methods. Either the gel may be transferred to a weakly acidic solution or to a solution containing an excess of a substance which forms strong silver complexes. The time needed in the stopping solution is short, in the order of 2-5 minutes, whereafter the gel can be evaluated. If the gel is to be preserved by drying, glycerol may be added to the stop solution to prevent the gel from cracking. Equilibration with glycerol will then take a longer time (10-20 minutes) than that needed to stop the development. The silver staining method according to the present invention may be used with all common electrophoretic methods applied to separate biomolecules. Neither SDS, high urea concentrations or any of the substances normally used in electrophoretic buffers will interfere with the method. The sensitivity is high enough to allow the method to be used for fast visualisation of DNA sequencing fragments electrophoretically separated in the presence of urea. Carrier ampholytes used in connection with isoelectric focusing do not either interfere with the method, but in order to obtain a high sensitivity after electrofocusing, the acid used for fixation in the combined fixation- impregnation solution should preferably be trichloroacetic acid or a mixture of trichloroacetic acid and sulphosalicylic acid. It is solely when the electrophoretic separation is carried out under reducing conditions generated with thiol-containing compounds, like mercapto- ethanol or dithiothreitol, that a pre-wash may be necessary. In SDS electrophoresis, large amounts of mercaptoethanol or dithiothreitol are added to the gel with the protein-containing sample. These thiol-containing compounds have to be eliminated from the gel prior to silver impregnation. If the buffer system described by Laemmli (Laemmli, U. K. (1970) Nature (London) 277. 680), or any of the buffer systems described in our International patent application PCT/SE95/01428 is used, this is done in connection with the electrophoresis as the thiol-containing compounds with these discontinuous buffer systems are transported with the front. Silver staining of these types of gels according to the method of the present invention without pre-wash result in the front appearing as a sharp black line in the stained gel. To have a permanent record of the position of the front is normally an advantage in SDS electrophoresis. With continuous buffer systems, as well as with discontinuous systems using borate, taurine or tricine, mercaptoethanol will be present in the gel at the end of the electrophoretic experiment. Mercaptoethanol precipitates silver, and therefore the gel region in which the thiol is present will become heavily stained. If proteins of interest are present in the same area, mercaptoethanol has to be eliminated by an initial wash. This initial wash is then done in a solution containing a high concentration of alcohol (30-50 %) and normally the same strong acid as that used later in the combined fixation-impregnation solution. In spite of the low pH used in the fixation- impregnation step, the compounds constituting the electrophoretic buffer may still to some extent form silver complexes. If the buffer concentration in the gel is high and the volume ratio of fixation-impregnation solution to gel is of the order of 10, fairly high silver concentrations (4-10 grams/litre) may be needed. Silver is expensive and in order to minimise the silver consumption, a pre-wash may in such cases be a preferred option. Generally, a short pre-wash (2-5 minutes) will suffice to reduce the required silver concentration in the fixation- impregnation solution to 1-2 grams/litre. As apparent from the above, silver staining according to the present invention normally requires only three steps: (1) fixation-impregnation; (2) development and (3) stop of development. The total time needed for a certain silver staining method will heavily depend on (i) the thickness of the gel, (ii) whether the gel is bound to a plastic foil, (iii) the polyacrylamide concentration, (iv) the temperature used, and (v) the sensitivity needed. Generally, the presently described method will use fewer solution changes than any of the prior described staining methods. With comparable gels, staining temperatures and resulting sensitivity, the method of the invention will save approximately 40-50 % of the time required in comparison with the earlier described "fast" silver staining methods. In the following, the invention is illustrated by some non-limiting Examples. EXAMPLE 1 Commercially available pre-cast gels (PhastGel® homogeneous 12.5, Pharmacia Biotech AB, Uppsala, Sweden) on plastic backing were utilised. The size of these gels was 43 x 50 x 0.45 mm. At the start of the electrophoresis the gels contained 0.112 M Tris-acetate, pH 6.4, and the gels were run with PhastGel® SDS buffer strips (Pharmacia Biotech AB, Uppsala, Sweden) containing 0.2 M Tris-tricine, pH 8.1, and 5.5 grams SDS/litre. The sample used was a commercial DNA size marker (Hae III digest of bacteriophage φX 174) . The digest was diluted to 8, 4, 2, 1, 0.5 and 0.25, respectively, μg DNA/ ml, and 1 μl of each dilution was applied to the gel. Staining was done with the development unit of the PhastSystem™ (Pharmacia Biotech AB) , which delivers 80 ml solution in each step, with the following solutions, temperatures and times: Fixation- 2 grams of 50°C 10 min impregnation silver/litre; 1% strong acid Wash H O 20°C l min Development 25 grams of 20°C 4 min Na^CO--,/litre; 1 ml 37% form¬ aldehyde/1itre; 8 g a^CO-^/litre Stop-preserve 14.6 grams of 50°C 10 min Na^EDTA/litre; 10% glycerol Figs. 2-4 show photographs of resulting silver-stained gels with perchloric acid, sulphosalicylic acid and benzenesulphonic acid, respectively, as strong acid in the fixation-impregnation step. These results may be compared with Fig. 1, which shows an identical gel silver stained according to the protocol of Bassam et al . , supra. No silver stained bands appeared when the strong acid was excluded from the fixation-impregnation solution, neither did any bands appear when phosphoric acid was used as acid in the fixation-impregnation solution, presumably due to the formation of a silver-phosphate complex. Replacement of the strong acid with 1 % acetic acid or 1 % lactic acid resulted in very faintly stained bands (Fig. 5) . EXAMPLE 2 This example illustrates the need of a pre-wash in cases where the gel after completed electrophoresis contains compounds with thiol groups. Gels and running conditions used are as in Example 1. The DNA digest was applied to four lanes using dilutions giving 2, 1, 0.5 and 0.25 μg DNA/ml, respectively. 1 μl of a dilution series of a commercial protein molecular weight marker mixture was applied to the remaining four lanes. The protein mixture was solubilised in 0.0625 M Tris-Cl, pH 6.8, containing 5 % mercaptoethanol, 2 % sodium dodecyl sulphate and 10 % glycerol. The approximate protein concentrations applied were 25, 12.5, 6 and 3 μg, respectively, of each protein per ml sample solution. The silver staining was either run as in Example 1 or alternatively a 10 minutes pre-wash (20 °C) preceded the fixation-impregnation step in the procedure used in Example 1. The pre-wash was performed with a solution containing 40% ethanol and 1 % strong acid. The same strong acid was used in the pre-wash and in the combined fixation-impregnation step. Figures 6, 8 and 10 show the resulting silver stained gels without pre-wash and with perchloric acid, trichloroacetic acid and nitric acid, respectively, in the fixation-impregnation step. Figures 7, 9 and 11, respectively, show the same experiments repeated with a pre-wash to get rid of the mercaptoethanol left around the sample application point. EXAMPLE 3 This example presents results with the staining method according to the present invention for vertical electrophoresis gels with and without, respectively, a high concentration of urea in the gel. Conventional equipment for casting and running vertical slab gels was utilised. 0.75 mm thick gels were cast between a notched and a plain glass plate of the size 105 x 100 mm. The separation part of the gel was 80 mm high and polymerised with persulphate and TEMED from a solution containing 7.5 g acrylamide/100 ml, 0.225 g bisacrylamide/100 ml, 0.375 M Tris, 0.0625 M Cl ~ , and for some of the gels 7M urea. On top of the separation gel, stacking gels were cast containing 4.0 g acrylamide, 0.12 g bisacrylamide, 0.5 M Tris-Cl, pH 6.8, and when urea was present in the separation gel, 7 M urea. The gels were run with an electrode solution (common to anode and cathode) composed of 0.025 M Tris and 0.192 M Cl ~ . The samples used were Hae III and Hinc II digests of bacteriophage φX 174 diluted to 4, 2, 1, 0.5 and 0.25 μg DNA/ml, respectively, and 3 μl of the dilutions were applied pro sample lane. Figures 12 and 13 show urea- containing gels using perchloric acid and naphthalenedisulphonic acid, respectively, as strong acid according to the present invention. Figure 14 shows a gel without urea where perchloric acid was used as strong acid. Figure 15 also shows a gel without urea and stained with perchloric acid as strong acid. In the latter gel, however, the Tris-chloride/Tris-glycine buffer was replaced by a Tris-borate-EDTA buffer. The following scheme was used for staining, all steps being performed at room temperature: Fixation- 4 grams of 20 min impregnation silver/litre; 1 % strong acid; Wash H^O 1 min Development 25 grams of 10 min Na^CO-^/litre; 1 ml 37 % form¬ aldehyde/litre; 8 g Na^S ? 0-^/litre Stop-preserve 14.6 grams of 10 min NapEDTA/litre; 10 % glycerol EXAMPLE 4 A silver staining kit contains: - 750 ml fix and silver solution [0.6% (w/w) benzenesulphonic acid, 0.2% (w/w) silver nitrate aqueous solution] ; - 10 x 75 ml developer solution [2.5% (w/w) sodium carbonate; 100 μl formaldehyde and 100 μl sodium thiosulphate/100 ml developer added just prior to use] ; - 750 ml stop and preserving solution [1% (w/w) EDTA, 10 % (vol. /vol.) glycerol]; - 1 ml formaldehyde; and - 1 ml 2% (w/w) thiosulphate aqueous solution. An electrophoretic gel (PhastGel®, Pharmacia Biotech AB) is treated with the above silver staining kit in the following way: 1. Impregnate the gel with the fix and silver solution for 10 min at 50°C; 2. wash in distilled water for 1 min at 25°C; 3. treat with developer solution for 4 min at 25°C; and 4. treat with stop and preserving solution for 10 min at 50°C. Optionally, the kit additionally contains: - 750 ml fix solution [0.6% (w/w) benzenesulphonic acid aqueous solution, optionally containing 40% (vol. /vol.) ethanol]. In this case, steps 1 to 4 above are preceded by the step of impregnating the gel with the fix solution for 8 min at 50°C. Table 1 Procedures for silver staining. Step Procedure according to Procedure according to Merril et al Bassam et al 1. Fix 10% acetic acid; 45 min 10% acetic acid; 20 min 2. Wash 10% ethanol; 5 min 3. Pre-treat K2Cr 2 θ7 (1 g/litre) , 0.2 ml HNO3/litre; 5 min 4. Rinse water; 3 times 2 min water; 3 times 2 min 5. Impregnate gNθ3 (2 g/litre); 30 min AgNOβ d g/litre), 1.5 ml 37% HCOH/litre; 30 min 6. Rinse water; 20 s (water; 20 s, optional) 7. Develop Na C03 (30 g/litre), 0.5 ml Na 2 C0 3 (30 g/litre) , 37% HCOH/litre; 5-10 min 1.5 ml 37% HCOH/litre; Na2S2θ3x5H 0 (2 mg/litre) 2-5 min 8. Stop 10% acetic acid; 5 min 10% acetic acid; 5 min

Claims: CLAIMS 1. A method of staining biomolecules present in or on a support medium, which method comprises fixing the biomolecules to the support medium, treating the support medium with a silver solution, and developing the thus- treated support medium with a reducing agent, characterised in that fixing of the biomolecules and treatment with silver solution are performed in a single step by impregnating the support medium with a silver solution containing silver ions and a strong acid which has a pK value lower than 3 and which at the impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution. 2. The method according to claim 1, characterised in that said strong acid is an inorganic acid, a carboxylic acid substituted with chloro or fluoro at its α-carbon or an organic acid containing a sulphonic group. 3. The method according to claim 2, characterised in that said strong acid is selected from nitric acid, hydrofluoric acid, chloric acid and perchloric acid. 4. The method according to claim 2, characterised in that said strong acid is selected from trichloroacetic acid and trifluoroacetic acid. 5. The method according to claim 2, characterised in that said strong acid is selected from methanesulphonic acid, benzenesulphonic acid, naphthtalenedisulphonic acid and sulphosalicylic acid. 6. The method according to any one of claims 1 to 5, characterised in that the concentration of said strong acid in said solution is between about 0.004 and about 1 M, preferably between about 0.01 and about 0.4 M. 7. The method according to any one of claims 1 to 6, characterised in that said solution after equilibration with the support medium has a pH value between about 0.2 and about 3, preferably between about 0.7 and about 2.5. 8. The method according to any one of claims 1 to 7, characterised in that said solution contains between about 0.4 and about 25 grams of silver/litre, preferably between about 1 and about 5 grams of silver/litre. 9. The method according to any one of claims 1 to 8, characterised in that said solution additionally contains up to 60 % by volume of alcohol, preferably selected from methanol, ethanol and propanol. 10. The method according to any one of claims 1 to 9, characterised in that said solution additionally contains a sensitiser. 11. The method according to any one of claims 1 to 10, characterised in that the method comprises the step of treating the support medium with a strong acid as defined in claim 1 prior to treating the support with the silver solution. 12. The method according to any one of claims 1 to 11, characterised in that said support medium is a gel . 13. The method according to any one of claims 1 to 12, characterised in that said biomolecules are selected from proteins, peptides and nucleic acids. 14. A silver staining kit for carrying out the method according to any one of claims 1 to 13 , characterised in that the kit comprises: (i) a fixing and silver solution comprising an acid having a pK value lower than 3 and which at the silver impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution, and a silver salt; (ii) a reducing agent comprising formaldehyde and, optionally, a thiosulphate; and (iii) a stop solution. 15. The silver staining kit according to claim 14 for carrying out the method of any one of claims 11-13, characterised in that the kit additionally comprises a fixing solution containing an acid having a pK value lower than 3 and which at the silver impregnation conditions can neither form strong complexes with the silver ions nor precipitate silver from the silver solution.

Inventors: BJELLQVIST BENGT, OLSSON INGMAR, BJELLQVIST, BENGT, OLSSON, INGMAR
Applicants: BJELLQVIST BENGT, OLSSON INGMAR, PHARMACIA BIOTECH AB, BJELLQVIST, BENGT, OLSSON, INGMAR
Application year: 1996
Main classification: G01N1
IPC codes: G01N1, G01N27, G01N33