JPS6235262A - Detection of protein or nucleic acid with high sensitivity - Google Patents

Abstract

PURPOSE:To detect the protein or nucleic acid separated by electrophoesis with high sensitivity by incorporating the compd. expressed by the specific general formula into a compd. which forms a dye by oxidation coupling reaction. CONSTITUTION:The protein or nucleic acid in a base such as gelatin or a gar gel separated by the electrophoresis is immersed in an aq. soln. of the metallic salt of silver, etc. and is dyed by binding the same with the metallic ions. An intensifier such as chlorous acid is acted to the resultant metallic nuclei in the presence of the compd. which forms the dye by the oxidation coupling reaction to form the detectabe compd. on the metallic nuclei. The compd. which forms the dye by the oxidation coupling reaction contains the compd. expressed by the general formula shown in the formula. In the formula, X is a univalent substituent which eliminates in the stage of the oxidation coupling reaction, n is 1-6 integer, R is a hydrogen atom or univalent substituent, each R may be the same or different when n>=2.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a highly sensitive method for detecting biological components electrophoretically separated in a support. More specifically, the present invention relates to a method for detecting even minute amounts of biological components such as proteins or nucleic acids separated by electrophoresis in a support.

[Conventional technology]

Proteins in body fluids such as plasma, urine, brain, cerebrospinal fluid, amniotic fluid,
For detection and identification of nucleic acids such as DNA and RNA,
Support-based electrophoresis methods have been widely used and have made significant contributions to research in the fields of biochemistry, biology, and clinical medicine.

Usually, the support is gelatin, agar gel, F paper,
Yumpun gel, cellulose acetate membrane, polyacrylamide gel, agarose gel, etc. are used, and electrophoresis methods include gel electrophoresis, 8DS electrophoresis, isoelectric focusing, immunoelectrophoresis, etc. Two-dimensional electrophoresis has also made it possible to separate complex protein mixtures such as plasma proteins.

As a method for detecting electrophoretically separated proteins,
Conventionally, this was done by dyeing with dyes.

The most commonly used dye is Kuumatsu Sieve IJ.
Liant blue, and black,
Crystal violet, fast green, etc. are used. In addition, electrophoretically separated DNA and RNA
were stained with ethidium bromide.

However, these staining methods have low detection sensitivity, so when analyzing samples with low protein concentrations such as urine or brain cerebrospinal fluid, a concentration operation is required before electrophoresis, which requires additional analysis procedures. This method is complicated, and also has the disadvantage that it involves changes in components during concentration.

In addition, from the viewpoint of highly sensitive detection, detection is performed by labeling with radioisotopes and is used in autoradiography, but since radioactive substances are handled, there are risks and complications in experimental operation. In addition to this, it is difficult to say that the technology is easy to implement, such as the issue of isotope disposal.

In recent years, silver staining, for example, has been developed as a highly sensitive detection method for proteins, nucleic acids, etc. separated by electrophoresis.

The steps of the silver staining method mainly include fixing separated proteins or nucleic acids in a support, immersing them in a silver ion or silver complex ion solution to bind them to silver, and treating them with a reducing agent. Become. For example, clinical
Chemistry Acta (C1inicC11nical
Che Acta) Vol. 38, pp. 465-467 (1972), Analytical Biochemistry
- (Analytical Biochemistry
stry ) No. 9 8 Volume tube 251 to 237
Page (1979), Electro 7 Oresis (1! ilea
trophoresig) Vol. 2, p. 135 and No. 1
41 (1981), and Volume 3, pp. 17-23 (1981).
There are various methods described in the literature such as "Biochemistry", Vol. 52, pp. 411-413 (1980), "Clinical Examination", Vol.・Nucleic acids/enzymes] Volume 72, Nos. 1277-1279
There are reviews such as Page (1982).

[Problem that the invention seeks to solve]

Although these silver staining methods have higher sensitivity than the above-mentioned detection methods using dye staining, they still cannot be said to have sufficient detection power.

The purpose of the present invention is to provide biological components separated by electrophoresis,
In particular, it is an object of the present invention to provide a detection method that enables highly sensitive detection after staining proteins and nucleic acids using a metal staining method.

[Means for solving problems]

To summarize the present invention, the present invention relates to a method for highly sensitive detection of separated proteins or nucleic acids, and includes a step of staining a protein or nucleic acid in a support separated by electrophoresis using a metal staining method, and In the presence of a compound that forms a dye by a monooxidative coupling reaction on the metal nucleus,
1. In a method for detecting a separated protein or nucleic acid that includes each step of forming a detectable compound on the metal core by applying an intensifying agent, the compound that forms a dye by the oxidative coupling reaction is The following general formula I: (wherein, In the above case, each R may be the same or different).

The inventors of the present invention have conducted extensive studies on the means described in Japanese Patent Application No. 18A211/1982, and have found that a pigment-forming compound containing the compound represented by the above general formula is used in combination with an intensifying agent. As a result, they discovered that this staining method allows for highly sensitive detection, and that the outflow of dye on a single staining spot is extremely small.

The process of staining by the metal staining method includes the steps of separating and fixing proteins or nucleic acids in a support, immersing them in an aqueous solution of metal salts to bond with the metal ions, and treating with a reducing agent. It includes a process.

The fixation step can be performed using glutaraldehyde, formaldehyde, paraldehyde, trichloroacetic acid, acetic acid, lead, osmium oxide, or the like. Generally, a mixed solution of acetic acid and methanol and/or ethanol and water is widely used.

Silver salts, gold salts, platinum salts, palladium salts, and iridium salts can be used as the metal salts used in the metal staining method in the method of the present invention, and specifically, silver nitrate, ammoniacal silver nitrate, silver oxalate, and dichloride Examples include gold acid, chloroplatinic acid, chloropalladic acid, chloroiridic acid, and the like. As a metal salt,
A water-soluble metal salt containing at least one metal selected from the group consisting of silver, platinum, gold, palladium, and iridium is used. Preferred are water-soluble silver salts that are readily available and inexpensive.

Reducing agents used in the reduction process include organic compounds such as formaldehyde, paraformaldehyde, hydroquinone, phenidone, pyrogallol, p-phenylenediamines, and p-aminophenols, and metals such as iron, tungsten, vanadium, molybdenum, and nickel. There are compounds. Formaldehyde and/or rose formaldehyde used in the silver staining method known in the above-mentioned literature is preferred.

In the present invention, the compound that forms a dye through an oxidative coupling reaction refers to a compound that forms a pigment after a certain compound undergoes an oxidation reaction.
It refers to a group of compounds in which the oxidized product further undergoes a coupling reaction with a certain coloring agent (referred to as a coupler) to form a dye.

After staining with a metal, when an intensifying agent is applied in the presence of a compound that forms a pigment through an oxidative coupling reaction, a pigment is formed on the metal nucleus, and as a result, proteins and nucleic acids can be stained with the metal plus pigment. can.

As an example of the intensifying agent referred to in the present invention, US Pat. No. 467
4.490, WJ 7/i 5.890, 3,776.730, etc. Peroxides such as hydrogen peroxide, perborate, percarbonate, etc. Examples include inorganic peroxides such as salts and persulfates, and organic peroxides such as benzoyl peroxide. Also, 765 to U.S. patent w.
．． No. 891, No. 824652, No. 4854.
? Examples include cobalt [phase] complexes described in the specifications of No. 07, No. 474a138, and JP-A-52-7728. Although almost all Lewis bases can be used as ligands for cobalt complexes, particularly useful ligands are:
Ethylenediamine (on), diethylenetriamine (d
ien),) Liederentetramiy (trian)
, Ammy (11BB), nitrate, nitride, azide, chloride, thiocyanate, inthiocyanate, water, carbonate, ethylenediaminetetraacetic acid, and the like. For example, the cobalt complexes shown below can be used.

(''(NHa)s Ha') 1% ('Q(
NHs)s (!0x)X 1(”(NHs)s'/)
X% (CO(NHs)aCon)X-.

(co(on),)X
)! .

Trans-(Co(on)IC#(N(119))!
.

yx-(Co(en)t(Is)t)X
%-/X-(00(an), (NHa)N,)X,
fi-(00(an),07.)X.

Tryx-(Co(an), C/,)χ, ((3
0(8n)t(8C!N)t)X 5(co(an)
, (wa8), :]! , ((!o(trlsn
)1)! .

(Co(trisn)t(en))! , (Oo(tr
ien) (8n), )! .

((5m), co-o-o-ao(yBa)s )X
%(ao(en), (d1+1n))X (where X represents one or more anions necessary to neutralize the charge). 1977
Examples include chlorous acid, chlorite, bromous acid, bromite, and chlorine dioxide water described in No. 53826 and No. 51-99022. Also, JP-A-52-7
6925, such as N-/% lokenphthalimide, N-halogen succinimide, N-halogen saccharin, chloramine-B, and chloramine-T.

In addition, transition metal complexes of group Vb of the periodic table described in JP-A-52-79928, such as Nag [
:'VO(0!)(0tOa)t)zo,Ka
(V(OxMC!toa)) &01Ka (NbO(O
s) JaROa) z) HtO and the like. Further, molybdenum and tungsten complexes described in JP-A-55-5626, for example, Ka [140a (
Ox 04)t (Hz O)t ), Nag (M(
0ri0CZa), Ka CMOs ((c&C!o
o)t NcHa 011(CL coo)t ) M
ow ) (where y represents MO or W), and the like.

Further, polyvalent iodine compounds described in JP-A-52-73731 can also be used.

Furthermore, JP-A-52-20025 and JP-A No. 52-304
The method described in No. 50 specification, that is, Oo
(Ell) complex and hydrogen peroxide may be used in combination.

Compounds that form a dye by an oxidative coupling reaction as used in the present invention, that is, a group of compounds in which a certain compound undergoes an oxidation reaction and then the oxidized product further undergoes a coupling reaction with a certain coupler to form a dye component. For example,
Written by T. H. James8 [The Theory of the Photographic Process J]
(The Theory of the Photographer
aphic Proceea) (4th edition) Chapters 11 and 12 for details.

This will be explained in detail below. As the compound that undergoes the oxidation reaction, for example, phenylenediamine-based or aminopyrazolone-based so-called photographic color developing agents are used.

However, heterocyclic hydrazines can also form dyes through oxidative coupling reactions.

As the phenylenediamine-based color developing agent, an aromatic primary amine color developing agent is preferable, and is disclosed in US Pat. ,
P-aminophenol developing agents in the form of onium salts as described in US Pat. No. 791.827, dye developers as described in US Pat. Specific examples of p-phinidine diamine color developing agents include 4-amino-N, N-diethylaniline, 3-methyl-4-ami2N, N-diethylaniline, and 4-amino-N-ethyl. -N-β-hydroxyethylaniline, 3-methyl-
4-amino-N-ethyl-N-β-hydroxyethylaniline, 3-methyl-a-7 minnow N-ethyl-N-β
-methanesulfonamidoethylaniline, 3-methyl-
4-amino-N-ethyl-N-β-methoxyethylaniline, 3-β-methanesulfonamidoethyl-4-amino-N, N-diethylaniline, 3-methoxy-4-amino-N-ethyl-N- β-hydroxyethylaniline, 5-methoxy-4-amino-N-ethyl-N-β-methoxyethylaniline, 3-acetamido-4-amino-N,N-diethylaniline, 4-amino-N,N-dimethyl Aniline, N-ethyl-N-β-[β-(β-methoxyethoxy)ethoxy]ethyl-3-methyl-4-
Aminoaniline, N-ethyl-N-β-(β-methoxyethoxy)ethyl-3-methyl-4-aminoaniline and salts thereof such as sulfate, hydrochloride, sulfite, p-toluenesulfonate, etc. .

In addition, these color developing agents are
As described in Publication No. 0, black and white developing agents such as phenidone derivatives may be used in combination.

Specific examples of aminopyrazolone color developing agents include 4-aminoantipyrine and the like. Specific examples of the heterocyclic hydrazines include 3-methyl-2-benzothiazoline hydrazoy and the like. These oxidized compounds are oxidized on the metallic silver core by the action of an intensifying agent, and the oxidized product reacts with a photographic fogger that has been previously present in the support or in the processing solution. Can form pigments.

As the above-mentioned coupler, a compound represented by the above-mentioned general formula I is used in the present invention.

This will be explained in detail below. Examples of monovalent substituents that are removed during the oxidized cup 9 fuf reaction represented by X include
-rogen atoms (e.g. fluorine atom, chlorine atom, bromine atom, iodine atom), alkoxy groups (e.g. methoxy group, ethoxy group, n-propoxy group, n-butoxy group, n
-hexyloxy group, etc.), aryloxy group (e.g., phenoxy group, naphthyloxy group, etc.), alkylthio group (e.g., methylthio group, etherthio group, n-propylthio group, n-butylthio group, n-hexylthio group, etc.), arylthio group (e.g., phenylthio group, naphthylthio group, etc.), alkylsulfonamide group (e.g., methanesulfonamide group, ethanesulfonamide group, etc.), arylsulfo/amide group (e.g., evabenzenesulfonamide group)
, an acyloxy group (eg, 7 acetyloxy groups, 7 propionyloxy groups, 7 benzoyloxy groups, etc.), an arylazo group (eg, a phenylazo group, a naphthylazo group, etc.), and a sulfo group.

These monovalent substituents represented by X may further include other substituents,
For example, halogen atoms, hydroxy groups, carboxy groups,
It may be substituted with a sulfo group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an amine group, etc., such as an isopropoxy group, a 2-hydroxyethoxy group, a 2-methoxyethoxy group, a carboxymethoxy group, 1 -carboxybutyloxy group, benzyloxy group, 2-chloroethoxy group, 2-(2-methoxyethoxy)ethkyne group, s-sulfopropyloxy group, 2p4,
6-) Lif'romophenoxy group, tolyloxy group,
4-carboquinphenoxy group, '-(L''-dimethylamino)phenoxy7 group, 2-hydroxyethylthio group,
2-ethoxyethylthio group, carboxymethylthio group,
2-carboxyethylthio group, benzylthio M, 1.2
-dicarboxyetherthio group, 2-carboxyphenylthio group, 4-hydroxybenzenesulfonamide group, 4
-Methoxyphenylazo group, 2-hydroxyethylthio group, 2-chlorophenylazo group and the like.

Examples of the monovalent substituent represented by R include a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), an alkyl group (for example, a methyl group, an ethyl group,
n-propyl group, n-butyl group, etc.), aryl group (e.g., phenyl group, etc.), alkoxy group (e.g., methoxy group, ethoxy group, n-propyloxy group, n-bunaloxy group, etc.), aryloxy group ( For example, phenoxy group, etc.), carboxy group, hydrococo group, sulfonic acid, carbamoyl group, acylamino group (e.g., acetylamino group, propionylamino group, benzoyl group, etc.), alkoxycarbo△2/' group (e.g., t, methoxycarbonyl group, ethoxycarbonyl group, etc.), aryloxycarbon2/14 (sidelight! d', phenoxycarbonyl group, etc.), amino group, nitro group, etc. These include alkyl group, aryl group, alkoxy group, Aryloxy group, carbamoyl group,
An acylamino group, an alkoxycarbonyl group, an aryloxycarbonyl group, or an amino group may further include any substituent such as an alkyl group, an aryl group, an alkoxy group, an aryloxy group, a halogen atom, a hydroxy group, a carboxy group, a sulfo group, or an amino group. It may have.

More specific examples of the compound represented by the above general formula 1 include (1) 4-chloro-1-naphthol (2) 2,4-dichloro-1-naphthol (3) 4-bromo-1-naphthol 7
Toll (4) 4-methoxy-1-su7 Toll (5)
'4-Ethoxy-1-naphthol (6) 4- (n
-ptoxy)-1-su7toll (7) 4- (2-
(chloroethoxy)-1-naphthol (8) 4-sulfo-1-naphthol (9) a-(2-methoxyethoxy)-1-naphthol 4-methylthio-1-naphthol α-4-7enyltheo 1- naphthol (6
) 4-phenylazo-1-naphthol (to) 4-(2
-Hydroxyphenylazo)-1-naphthol α4a
-(a-methoxyphenylazo)-1-naphthol (
g) 4,5-dimethoxy-1-naphthol Q$ 4-
Ethanesulfonamide-1-naphthol αf) a-(
2-aminophenyl 7so) -2-propyl-1-naphthol α→4-[2-hydroxy-5-((1-isopropyl)amino]propoxy]-1-naphthol α
4,7-Simethoxy-2-methoxymethyl-1-su7
Toll 4-chloro-2-dimethylcarbamoyl-1
-naphthol (2) 4-(1-carboxybutoxy)
-1-naphthol (a) 4-chloro-2-acetylamino-1-naphthol and the like can be mentioned.

In the detection method of the present invention, it is preferable that the compound that forms a dye by oxidative coupling reaction is impregnated into the support. In this case, a method of immersing the support in a solution of a compound that forms a dye by an oxidative coupling reaction,
Alternatively, there is a method of spraying the solution.

As a method for causing the reinforcing agent to act on the metal core, there are methods such as dipping or spraying using one solution as described above.

Further, the compound that forms a dye through an oxidative coupling reaction and the intensifying agent can be impregnated into the support in one operation in the same solution, thereby shortening the processing operation.

In the method of the present invention, the processing steps for staining electrophoretically separated proteins or nucleic acids usually include (1) electrophoresis step - immobilization step - binding step with metal ions - reduction step - intensification step (2) The process is basically an electrophoresis process, an immobilization process, a bonding process with metal ions, and a reduction/enhancement process. Furthermore, a water washing step may be provided before and after each step after electrophoresis.

The above-mentioned reinforcing step means a step in which a reinforcing agent is applied to form a dye on a metal nucleus in the presence of a compound that forms a dye through an oxidative coupling reaction.

Solutions of compounds that form pigments through oxidative coupling reactions and solutions of intensifiers include alkaline agents such as caustic soda, caustic potash, soda carbonate, potassium carbonate, tribasic sodium phosphate, potash, potassium metaborate, and borax. , disodium or potassium hydrogen phosphate, sodium or potassium dihydrogen phosphate, sodium or potassium bicarbonate, boric acid, sodium or potassium acetate, sodium or potassium sulfate, and the like may be appropriately selected and contained.

Anti-capri agents can also be added to each of the above processing steps. As anti-capri agents, alkali metal halides such as potassium bromide, sodium bromide, potassium iodide, sodium iodide and organic anti-capri agents can be used. Examples of organic anti-capri agents include nitrogen-containing heterocyclic compounds (e.g. eva, hensitriazole, 6-nitrobenzimidazole, 5-nitrofindanol, 5-methylbenzotriazole, 5-nitrobenzotriazole,
5-chlorobenzotriazole, etc.), mercapto-substituted heterocyclic compounds (sidelight, 1-phenyl-5-mercaptotetrazole, 2-mercaptobenzimidazole, 2-mercapto-substituted heterocyclic compounds, etc.)
-mercaptobenzothiazole, etc.) mercapto-substituted aromatic compounds (such as, for example, thiosalicylic acid).

The amount added is in the range of 1 mg to 5 tons per 11 solutions.

In addition, solutions of compounds that form pigments through oxidative coupling reactions and solutions of intensifying agents include preservatives such as alkali metal sulfites, methanol, ethanol, isopropyl alcohol, acetone, benzyl alcohol, and diethylene glycol. Organic solvents such as, polyethylene glycol, quaternary ammonium salts, and development accelerators such as amines may be included as appropriate.

In order to stabilize the intensifying agent solution, organic phosphonic acids, polyvinylhyloritone, polyvinyl alcohol, or U7 amine oxides known in the photographic industry may be included.

As a detection method, visual detection is generally easy, and semi-quantitative or quantitative detection is possible by measuring the dye concentration.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The invention is not limited to these examples.

Example 1 Nature Vol. 227, p. 680 (
10% polyacrylamide gel (16cIR x 12cIIK), 1fi thick, based on the method described in (1970).
A gel was prepared, and 8D8-electrophoresis was performed using a molecular weight marker for electrophoresis (7 Almaqua) as a sample. Electrophoresis after electrophoretic separation
rophoresis) Vol. 2, p. 141 (1981
), staining was then carried out using the silver staining method. That is, the gel was mixed with a 50% methanol/10% acetic acid aqueous solution 2
00- for 30 minutes, then 15% methanol aqueous solution 200- for 3 times for 10 minutes each, 6% glutaraldehyde 200j for 30 minutes, 15% methanol aqueous solution, and further washing with water. Removal of sodium sulfate, etc. and immobilization of proteins were performed. Next, ammoniacal silver nitrate solution (α02N
Add 25% ammonia water 6- and 20% silver nitrate 8- to NaOH 160-, make 200- with water) for 10 minutes (2
After rinsing with water for 10 minutes, add 37% formalin [11m] to O,OO% citric acid 200-.
The gel was immersed for about 10 minutes and washed with water to obtain a silver-stained gel.

Next, high-sensitivity staining by pigment formation was performed using this silver-stained gel.

First, the gel was immersed in a solution (1) having the following composition for 5 minutes (at room temperature).

Liquid composition (1) (prepared immediately before use) Next, the gel was washed with water for 30 seconds, and the following composition was applied. l (2
).

Liquid composition (2) (prepared immediately before use) After immersion, color began to develop, and after 5 minutes, the gel was washed several times with water to obtain a dyed gel.

On the other hand, instead of 2,4-dichloro-1-naphthol in liquid composition (1), s,s-cyclo2-hydroxy-4-
A stained gel was obtained by performing the same treatment as above except that methylacetanilide was used.

As a result, when looking at ovalupmin (Oval+umin) as a standard, it was found that 10n? , 5nP, 1nf, α5nf, 0.1
When 5DEI-electrophoresis was performed with the addition of nf, a trace amount of 1nf of protein could be detected in each dye-stained gel, but when each stained gel was immersed in water and left for 3 days, 3,5 -dichloro-2-hydroxy-4-
In the gel stained with methylacetanilide, some dye flow was observed and the 1 nlF ovalpmin spot disappeared, but the protein spot stained with 2,4-dichloro-1-naphthol was maintained.

Example 2 In the same manner as in Example 1, a z5% polyacrylamide gel was created using a molecular weight marker for electrophoresis, and 8DS
- Separation was carried out by electrophoresis. Next, staining was performed using a silver staining method. That is, the gel was mixed with 50% methanol/1
0% acetic acid water soluble! The sample was immersed in 400 dK with shaking for 30 minutes, then immersed in a 15% ethanol aqueous solution AOOwAt for 15 minutes with shaking, and then washed with water to immobilize the protein. Next, 200-
After washing with water for 15 minutes, 35% formalin α1- was added. The gel was immersed in a 1% sodium carbonate aqueous solution with shaking for 10 minutes and washed with water to obtain a silver-stained gel. Furthermore, this gel was processed in the following additional steps.

A solution (4) K% gel having the following composition was immersed at room temperature with shaking.

Liquid composition (4) (prepared immediately before use) Color development started immediately after immersion, and after immersion for 10 minutes, the gel was washed several times with water to obtain a gel dyed according to the present invention.

As a result, when looking at ovalbumin as a standard, it was found that after serial dilution, each 10n? , 5nf, 1
nf, (L 5nf, α1nr added, EIDB
- Electrophoresis was performed on the stained gel, [L 5 n
A trace amount of protein j' (visual judgment) could be detected.

In this way, it has been found that high sensitivity detection is possible even when the compound that forms a dye through an oxidative coupling reaction and the intensifying agent (hydrogen peroxide) are used in the same solution, and it is preferable because the processing operation is simple. Ta. Furthermore, even when the gel stained according to the present invention was left immersed in water for 3 days, the formed dye did not flow out and the detected protein spots were maintained. The pigment formed was a deep blue color, and the color tone was easy to observe when visually detected.

Example 3 Plasma proteins were subjected to isoelectric focusing in the first dimension (4, 5
% polyacrylamide gel, pTl gradient 55~
9.5) Separate and make the second thick part, E? DB electrophoresis (10
% polyacrylamide gel, gel thickness 1) Separated. This gel was stained according to the silver staining method used in Example 2.

The silver-stained gel thus obtained was mixed into a solution (5) with the following composition.
The sample was shaken and immersed in water for 7 minutes at room temperature.

Liquid composition (5) (prepared immediately before use) CO potassium carbonate 2.5204-
Methoxy-1-naphthol 1.0 to ethanol 50-〇 water
150-050% hydrogen peroxide solution
Color began to develop immediately after immersion in ao sd. After 7 minutes, the gel was washed several times with water. In this manner, even in two-dimensional electrophoresis, highly sensitive detection was possible using the staining method according to the present invention. Even when this stained gel was immersed in water and left for 5 days, no dye leaked out and the detected protein spots were maintained.

Example 4 8% polyacrylamide gel (thickness CL711III)
and bacteriophage λ (C857sam).
7) DNA treated with EQQR restriction enzyme
The fragments were developed by electrophoresis in varying amounts and used in Example 2.
Silver staining was performed using the silver staining method shown in .

The 4 silver-striped gel thus obtained was mixed with a solution of the following composition (6
) K% Soaked at room temperature.

Liquid composition (6) (prepared immediately before use) After 2 minutes of immersion, 30% hydrogen peroxide water was added and immersed for an additional 7 minutes. Color development started immediately after adding hydrogen peroxide.

The gel was then washed several times with water to obtain a gel stained according to the invention. A DNA fragment of about 5nf could be detected.

Example 5 A pot-stained gel after 8D8-electrophoresis was prepared in the same manner as shown in Example 2, and then this silver-stained gel was treated with the solution (7) having the following composition according to the present invention. Two stainings were performed.

Liquid composition (7) (prepared just before use) After soaking for 10 minutes at room temperature, the gel was washed several times with water.

As a result, using ovalupmin as a standard, approximately (
It was found that trace amounts of L5nf protein could be detected and staining could be performed with high sensitivity. Further, even when this dyed gel was left immersed in water for 3 days, no outflow of the dye on the tarebacterial spots was observed.

Example 6 In the same manner as shown in Example 2, a silver-stained gel was prepared after 5D8 electrophoresis. Next, this silver-stained gel was immersed in a solution (8) having the following composition to form a dye.

Liquid composition (8) (prepared immediately before use) A dyed gel was obtained by washing twice with water. It was found that trace amounts of proteins could be stained with high sensitivity, and no dye leakage was observed on the spot.

Example 7 In the same manner as in Example 1, a 7.5% polyacrylamide gel was created using a molecular weight marker for electrophoresis, and 8D
8-Separation was performed by electrophoresis, and then staining was performed using the silver staining method by the treatment shown below. That is, 5 gels
Immerse in 0% methanol/10% acetic acid aqueous solution 400- for 20 minutes with shaking, then immerse in 15% ethanol aqueous solution 400-
After shaking and immersing in 00 vessel for 15 minutes, the protein was immobilized by washing with water. Next, it was immersed in silver nitrate aqueous solution 'ffL200dK with a concentration of 5 x 10''M/l, washed in two portions for 15 minutes each, and then added with 35% formalin α5-.
It was immersed in a 200% aqueous solution of sodium carbonate for 1θ minutes and water-stained to perform silver staining. No stained protein spots were observed in the gel obtained here (by visual inspection).

Further, the above gel was immersed in a solution (9) having the following composition, and subjected to an intensification treatment according to the present invention.

Liquid composition (9) (prepared immediately before use) After soaking for 10 minutes and washing with water several times, proteins that were not stained by silver staining were able to be dyed by the staining method of the present invention. .

Considering ovalbumin as a standard, it is serially diluted and placed in the groove of the concentrated gel at 100 nlF and 10 nlF, respectively. ,1nj,
When 5DS-electrophoresis was performed with the addition of alnr, 10 nIF of protein was stained in the gel subjected to the intensification treatment according to the present invention. In this way, one dyeing method according to the present invention is
Even in gels that were insufficiently stained by silver staining, it was found that trace amounts of protein could be detected by adding a simple intensification treatment.

〔Effect of the invention〕

As explained above, according to the method of the present invention, the staining concentration of electrophoretically separated proteins and nucleic acids is enhanced by adding a simple procedure of pigment formation to the staining method using the metal staining method. Sensitive staining could be performed. Furthermore, it has become possible to detect trace amounts of the above components that cannot be visualized using metal staining methods.

Claims (1)

[Claims] 1. A step of staining a protein or nucleic acid in a support separated by electrophoresis by a metal staining method, and the presence of a compound that forms a dye on the obtained metal nucleus through an oxidative coupling reaction. In the method for detecting isolated proteins or nucleic acids that includes each step of forming a detectable compound on the metal core by applying an intensifying agent, the compound that forms a dye by the oxidative coupling reaction is , the following general formula I: ▲Mathematical formulas, chemical formulas, tables, etc.▼...[I] (In the formula, X is a monovalent substituent that leaves during the oxidative coupling reaction, n is an integer from 1 to 6, R represents a hydrogen atom or a monovalent substituent, and when n is 2 or more, each R may be the same or different) Highly sensitive detection of isolated proteins or nucleic acids characterized by containing a compound represented by Method.