Silver staining method
BACKGROUND OF THE INVENTION 
     (i) Field of the Invention 
     This invention relates to silver stain and more particularly, to a method for staining a carrier, which bears thereon substances being detected, with silver in which the carrier is pretreated with an neutral or approximately neutral alcoholic solution comprising polyethylene glycol or a polyoxyethylene alkylphenol and subsequently treated with a silver staining solution of a specific type. 
     (ii) Description of the Prior Art 
     At present, various detecting methods have been put into practice, of which attention has been recently paid to silver stain because of its relatively higher sensitivity than other methods. The silver staining method has been widely used since it allows electrophoretic analysis, of samples having a low concentration of a substance to be detected such as, for example, proteins, nucleic acids, sugars, and lipids. Examples of such samples are urine, cerebrospinal fluid, etc. 
     A number of methods for silver staining have been reported (SEIKAGAKU Vol. 52, page 411, 1980, Protein Nucleic Acid and Enzyme Vol. 27, page 1277, 1982,  Electrophoresis Vol. 2, pages 141 and 135, 1981). However, all of the methods have the drawback that they require a long time to complete staining, and complicated operations are necessary because the procedure employed depends on the type of substance being detected. According to our experience, all the known staining methods also have the disadvantage of low reproducibility. 
     For instance, according to Poehling et al (Electrophoresis Vol. 2, 141, 1981), after electrophoresis, the carrier is fixed with a 50% methanol/10% acetic acid solution for 30 minutes, thereby suppressing coloration of the background of the carrier, and subsequently fixed with a 6% glutaraldehyde solution to increase the stain sensitivity. However, these reagents should be removed by washing with methanol and water several times. These pretreatments therefore require a long time. 
     SUMMARY OF THE INVENTION 
     In order to overcome the drawbacks of the prior art, we have made intensive studies and as a result, have found that when a carrier is treated with an alcoholic solution comprising polyethylene glycol or a polyoxyethylene alkylphenol, the carrier shrinks and becomes easy to wash, resulting in reduction of washing time. Furthermore, it was found that when a silver staining solution is prepared which has specific ratios of silver nitrate; a compound of the formula R--NH 2 , wherein R--NH 2  represents ammonia or a primary amine; and a caustic alkali, it is possible to shorten the operation time and improve the reproducibility of staining. 
     According to the present invention, there is provided a silver staining method which comprises pretreating a carrier bearing thereon a substance to be detected with an alcoholic solution comprising alcohol and water and containing polyethylene glycol or polyoxyethylene alkylphenol; and treating the pretreated carrier with a solution comprising silver nitrate, a compound of the formula R--NH 2  (R--NH 2  has the same meaning as defined above) and a caustic alkali in ratios by molecular weight of 1:4.5-9.5:1.0-26.0. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The FIGURE is a band image obtained according to Example 3 in which a is a band image of BSA, and 1, 2, 3, 4, 5 and 6 are, respectively, images obtained in electrophoresis of 5 ng, 1 ng, 500 pg, 100 pg, 50 pg and 10 pg of BSA. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS 
     In order to carry out the method of the present invention, a carrier which has been subjected to electrophoresis by a usual manner (hereinafter referred to simply as gels) is immersed in an alcoholic solution containing polyethylene glycol or a polyoxyethylene alkylphenol (hereinafter referred to as the washing and fixing solution) for about 10 to 60 minutes, preferably about 25 minutes, thereby causing the gels to shrink. By the shrinkage, the water and the reagent are released from the gels. The alcohols used in the washing and fixing solution include lower alcohols and preferably linear or branched alcohols having from 1 to 4 carbon atoms. Examples of such linear or branched alcohols include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and mixtures thereof. Of theses, isopropanol, or a mixture of methanol and ethanol is preferred. These alcohols are conveniently used as an aqueous solution having a concentration of from 30 to 70 (v/v)%, preferably about 50 (v/v)%. 
     Polyethylene glycol suitable for the purpose of the invention has an average molecular weight of from 600 to 3500, preferably 1500 to 2500. The polyoxyethylene alkylphenol is represented by the formula, ##STR1## in which R&#39; represents a linear or branched alkyl having from 6 to 10 carbon atoms, and n is an integer of 7 to 10 which indicates the center of the distribution. Preferably, in the above formula, R&#39; is octyl and n is 9 which indicates the center of the distribution. The polyethylene glycol (PEG) or polyoxyethylene alkylphenol is generally used in a concentration of 0.05 to 5 (w/v)%, preferably about 2 (w/v)%, of the washing and fixing solution. 
     In the washing and fixing solution, if the concentration of alcohol is below 20 (v/v)%, the background of the gels is colored, making it difficult to confirm stained bands. On the other hand, when the concentration exceeds 90 (v/v)%, bands do not appear clearly. The concentration of polyethylene glycol or polyoxyethylene glycol below 0.01 (w/v)% is unfavorable because clear bands are not obtained. A concentration over 5 (w/v)% does not give any further effect. 
     The gels pretreated with the washing and fixing solution are subsequently immersed in and treated with a silver staining solution for 10 to 30 minutes, preferably about 15 minutes. The silver staining solution used for the above treatment is a mixture of silver nitrate, a compound of the formula R--NH 2  (R--NH 2  has the same meaning as defined before), and a caustic alkali in mixing ratios by molecular weight of 1:4.5-9.5:1.0-26.0. The treatment with the silver staining solution within the above mixing ratios permits staining with good reproducibility and a reduction of the operation time. It will be noted that when the mixing ratio of the R--NH 2  compound is below 4 and the mixing ratio of the caustic alkali is over 28 based on the molecular weight, considerable unfavorable precipitation of silver oxide takes place. On the other hand, when the mixing ratio by molecular weight of ammonia is over 10 and the mixing ratio of caustic alkali is below 0.5, it is difficult to stain the gels. 
     Compounds of the formula, R--NH 2 , usable in the present invention include, for example, ammonia, ethanolamine, propanolamine, isopropanolamine, butanolamine, isbutanolamine, tris(hydroxymethyl)aminomethane, methylamine, ethylamine, propylamine, butylamine, isobutylamine, etc. Examples of the caustic alkali include sodium hydroxide, potassium hydroxide, etc. 
     The gels treated with the silver staining solution are then immersed in a developer solution for about 10 to 15 minutes. If necessary, after treatment with the developer, the gel may be further immersed in a solution of 0.5 to 2 (w/v)% chloroauric acid, by which a clearer image is obtained. 
     The method of the present invention is conveniently applied to polymerized gels having a network structure, such as polyacrylamide gel, agar-agar, agarose, etc. 
     The method of the invention and conventional methods are summarized in Table 1. 
     
                                           TABLE 1                                 
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       Method of Invention                                                
                  Poehling et al Method                                   
                             Sammons et al Method                         
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Pretreat-                                                                 
       50% isopropanol +                                                  
                  50% methanol +                                          
                             50% ethanol +                                
ment Step                                                                 
       2% PEG     10% acetic acid                                         
                             10% acetic acid                              
       25 min.    30 min.    120 min. × 2                           
(Washing &amp;        15% methanol                                            
                             25% ethanol +                                
Fixing            30 min     10% acetic acid                              
Solution)                    60 min. × 2                            
                  6% glutaraldehyde                                       
                             10% ethanol +                                
                  30 min.    0.5% acetic acid                             
                             60 min. × 2                            
                  15% methanol                                            
                   ##STR2##                                               
Silver Ammoniacal silver                                                  
                  Ammoniacal silver                                       
                             Silver nitrate                               
Staining                                                                  
       nitrate solution                                                   
                  nitrate solution                                        
                             solution                                     
Step   silver nitrate 1                                                   
                  silver nitrate 1                                        
       ammonia 7  ammonia 9.2                                             
(Silver                                                                   
       caustic soda 4.3                                                   
                  caustic soda 0.34                                       
Staining                                                                  
       ratio by   ratio by                                                
Solution)                                                                 
       molecular wt.                                                      
                  molecular wt.                                           
       15 min.    15 min.    120 min.                                     
Washing                                                                   
       washing with water                                                 
                  washing with water                                      
                             washing with water                           
Step   5 min.     6 min.     10-20 seconds                                
Developing                                                                
       0.005% citric acid                                                 
                  0.005% citric acid                                      
                             3% caustic acid                              
Step   0.02% formalin                                                     
                  0.02% formalin                                          
                             0.009% sodium                                
(developer)                                                               
       15 min.    15 min.    boron hydride                                
                             0.28% formalin                               
                             10 min.                                      
                  washing with water                                      
                             0.75% sodium                                 
                  10 min. × 3                                       
                             carbonate                                    
                             60 min. × 2                            
                             0.75 sodium                                  
                             carbonate                                    
                             360 min.                                     
Total Time                                                                
       1 hour     4.1 hours  18 hours                                     
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     As will be seen from the above table, the method of the invention ensures formation of a clear image in about 60 minutes. Moreover, the same procedure may be applied to any substance contained in gels such as proteins, nucleic acids, sugars, lipids, etc. 
     The method of the invention has the following features: 
     (1) Neither glutaraldehyde, which is unstable as a fixing agent, nor acids such as trichloroacetic acid, acetic acid, etc which involve danger in handling are needed; 
     (2) No operation of removing the fixing agent is necessary; 
     (3) The pretreatment is simple; and 
     (4) High sensitivity and good reproducibility are obtained. 
     The present invention is described in detail by way of examples. 
     EXAMPLE 1 
     Gels were washed with washing and fixing solutions having different concentrations of alcohol and polyethylene glycol to allow determination of the presence or absence of bands and coloration on the background. The results are shown in Table 2. 
     Sample 
     A gel was obtained by subjecting a Hind III fragment of λ phage DNA to electrophoresis using polyacrylamide gel. 
     Reagents 
     (1) Silver staining solution 
     Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water in ratios by molecular weight of 1:7:4.3 to make a total volume of 200 ml. 
     (2) Developer solution 
     0.05% citric acid, 37% formalin and distilled water were used to make 200 ml of a 0.02 v/v% formalin solution of 0.005 w/v% citric acid. 
     Operation 
     Each sample was immersed for 25 minutes in mixtures of isopropanol and polyethylene glycol 2000 having different concentrations indicated in Table 2, after which each sample was immersed in a silver staining solution for 15 minutes to cause the solution to penetrate into the gel. Thereafter, the sample was placed in a pan containing 200 ml of distilled water for washing. This procedure was repeated three times. Finally, the sample was immersed in a developer solution for 15 minutes with the result that a brown to black band image appeared. 
     
                       TABLE 2                                                     
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                  Evaluation*                                             
Concentration                                                             
           Concentration                                                  
                        Presence                                          
of Isopropanol                                                            
           of Polyethylene                                                
                        or absence                                        
                                  Background                              
(v/v) %    glycol (w/v) %                                                 
                        of bands  coloration                              
______________________________________                                    
0                       +         X                                       
10                      +         X                                       
30         2            ++        O                                       
50                      +++       O                                       
70                      +         O                                       
90                      -         O                                       
           0            +         X                                       
           0.01         +         O                                       
           0.05         +         O                                       
           0.1          ++        O                                       
50         1            +++       O                                       
           2            +++       O                                       
           4            +++       O                                       
           6            +++       O                                       
           10           +++       O                                       
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 *Evaluation Standard                                                     
 Presence or Absence of Bands                                             
 +++ Band image is very clear.                                            
 ++ Band image is clear.                                                  
 + Band image free of any practical problem obtained.                     
 ± Some problems are involved such as poor reproducibility, unclear ban
 image, etc.                                                              
 - No band image is obtained.                                             
 Background Coloration                                                    
 O No coloration.                                                         
 X Coloration.                                                            
 
    
     EXAMPLE 2 
     A solution of 2 g/l of silver nitrate was provided, to which different amounts of sodium hydroxide and ammonia were added to vary ratios thereof by molecular weight. The presence or absence of bands was checked and the optimal mixing ratios were determined. The results are shown in Table 3. The sample, developing solution and operation procedure were the same as in Example 3 described hereinafter. 
     
                                           TABLE 3                                 
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Ratio by M.W.                                                             
        Ratio by M.W. of Caustic Alkali                                   
of Ammonia                                                                
        0.53                                                              
           1.06                                                           
              2.13                                                        
                 4.25                                                     
                     8.5 12.75                                            
                             17                                           
                               21.25                                      
                                  26                                      
                                    29.75                                 
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3.15    ±                                                              
           ±                                                           
              ±                                                        
                 ±                                                     
                     ±                                                 
                         ±                                             
                             ±                                         
                               ±                                       
                                  ±                                    
                                    ±                                  
4.0     ±                                                              
           ±                                                           
              ±                                                        
                 ±                                                     
                     ±                                                 
                         ±                                             
                             ±                                         
                               ±                                       
                                  ±                                    
                                    ±                                  
4.73    ±                                                              
           +  +  ++  ++  +   + +  + ±                                  
6.3     -  +  ++ +++ +++ +++ + +  + ±                                  
7.88    -  +  +  +++ +++ ++  + +  + ±                                  
9.5     -  +  +  +   +   +   + +  + ±                                  
11.03   -  ±                                                           
              ±                                                        
                 ±                                                     
                     ±                                                 
                         ±                                             
                             ±                                         
                               ±                                       
                                  ±                                    
                                    ±                                  
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     The evaluation standard is the same as in Example 1. 
     EXAMPLE 3 
     Sample 
     A gel was obtained by subjecting bovine serum albumin to electrophoresis with sodium dodecylsulfatepolyacrylamide gel (hereinafter referred to simply as SDS-PAG). 
     Reagents 
     (1) Washing and fixing solution 
     Isopropanol and polyethylene glycol 2000 (by Furuka A. G., Switzerland) were diluted with distilled water to make a total volume of 200 ml with concentrations of 50% (v/v) and 1% (w/v), respectively. 
     (2) Silver staining solution 
     Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water to have mixing ratios by molecular weight of 1:4.8:1. The total volume was brought up to 200 ml. 
     (3) Developer 
     0.05% citric acid, 37% formalin and distilled water was used to make a total 200 ml of a 0.02 v/v% formalin solution of 0.005 w/v% citric acid. 
     Operation 
     Reagents (1), (2) and (3) were, respectively, placed in a cabinet-size pan. The sample was first immersed in the pan of reagent (1) for 25 minutes and removed from the pan, followed by immersing in the reagent (2) thereby causing the silver staining solution to penetrate into the gel. The sample was immersed in a pan containing 200 ml of distilled water and washed. This procedure was repeated three times. Finally, the washed sample was immersed in reagent (3) for 15 minutes to form a brown to black band image. 
     EXAMPLE 4 
     Sample 
     Same as used in Example 3 
     Reagents 
     (1) Washing and fixing solution 
     Methanol, ethanol and polyethylene glycol 2000 were diluted with distilled water to make a total volume of 200 ml with concentrations of 40% (v/v), 10% (v,v) and 2% (w/v), respectively. 
     (2) Silver staining solution 
     Silver nitrate, ammonia and sodium hydroxide were dissolved in distilled water to have mixing ratios by molecular weight of 1:7:4.3. The total volume was brought up to 200 ml. 
     (3) Developer 
     Same as used in Example 3. 
     Operation 
     The operation of Example 3 was repeated using the reagents indicated above, thereby obtaining a band image similar to the image of Example 3. 
     EXAMPLE 5 
     Sample 
     Same as used in Example 3. 
     Reagents 
     (1) Washing and fixing solution 
     Isopropanol and polyoxyethylene phenol (commercial name of Rhom Haas Co., Ltd., of U.S.A.: Nonizet P-40) were diluted with distilled water in concentrations of 50% (v/v) and 3% (v/v) to make a total volume of 200 ml. 
     (2) Silver staining solution 
     Silver nitrate, ammonia and sodium hydroxide were dissolved in ratios by molecular weight of 1:9.1:25.5 to make a total volume of 200 ml. 
     (3) Developer 
     Same as used in Example 3. 
     Operation 
     The procedure of Example 3 was repeated using the reagents indicated above, thereby obtaining a band image similar to the image of Example 3. 
     EXAMPLE 6 
     Sample 
     Same as used in Example 3. 
     Reagents 
     (1) Washing and fixing solution 
     Same as used in Example 3. 
     (2) Silver staining solution 
     Silver nitrate, tris(hydroxymethyl)aminomethane and sodium hydroxide were dissolved in distilled water in ratios by molecular weight of 1:5:1.0 to make a total volume of 200 ml. 
     (3) Developer 
     Same as used in Example 3. 
     Operation 
     The procedure of Example 3 was repeated using the reagents indicated above, thereby obtaining a band image similar to the image of Example 3.