JP2610493B2 - Silver staining method and silver stain kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to silver for the purpose of detecting biological components fractionated on a support carrier such as proteins, nucleic acids, sugars and lipids in electrophoretic analysis and the like. It relates to an improved method of dyeing.

[Conventional technology and its problems]

In the silver staining method, a support carrier containing a substance to be detected (hereinafter, referred to as “analyte”) is treated with an immobilizing agent, the analyte is immobilized, pre-treated, and then treated with a silver staining solution. This is a method of developing with a reducing agent, which enables analysis of low-concentration samples of test substances such as proteins, nucleic acids, sugars, lipids, etc., ie, serum urine, cerebrospinal fluid, etc., without performing a concentration operation. the study,
This is a useful analytical method widely used in clinical tests and the like.

Various methods of silver staining have been reported (Biochemistry 52).
Volume 411, 1980, Proteins, nucleic acids, enzymes 27, 1277, 1982
Electrophoresis, Vol. 135, p. 141, p. 141, 1981), all of which require a long time to complete the dyeing or require complicated operations, and the obtained silver stained images also have different dyeing properties depending on the substance to be dyed. The concentration sample has many problems such as not being completely stained (becoming a hollow state) and poor contrast due to an increase in background, and improvement has been desired.

[Means for solving the problem]

The present inventors have conducted intensive studies with the aim of shortening the operation time of the silver staining method, improving the dyeing ability and improving the contrast in order to solve such problems. It has been found that the addition of the compound can solubilize free silver ions on the support, prevent coloration of the support and improve the contrast of the dyed image of the developer, and completed the present invention.

That is, the present invention provides a method of treating a support carrier containing a test substance with an immobilizing agent to fix the test substance, pre-treating with a sensitizer, then treating with a silver staining solution, and then developing with a reducing agent. It is an object of the present invention to provide a silver dyeing method characterized in that a thiosulfate is contained in a reducing agent in a dyeing method, and a silver dye kit used for the method.

The reducing agent used in the silver staining method of the present invention may be any reducing agent having a silver ion reducing ability and containing a thiosulfate therein, but is preferably formaldehyde and citric acid. And the like. Examples of the thiosulfate include sodium thiosulfate and ammonium thiosulfate. Of these, sodium thiosulfate is preferred.

The sodium thiosulfate concentration in the reducing agent according to the present invention is:
0.000005W / V% or more may be sufficient, but especially 0.00002-0.00%
1 W / V% is preferred. When the reducing agent contains formaldehyde and citric acid, the formaldehyde concentration is generally 0.001 W / V% or more, preferably 0.01 to 0.04%.
W / V%, and the concentration of citric acid is 0.0005 W / V% or more, preferably about 0.002 to 0.01 W / V%.
Also, the weight ratio of formaldehyde to citric acid is 1: 0.25 ~
0.5 is preferred.

A development time of 5 to 10 minutes using this reducing agent is sufficient.

The method of the present invention can be carried out according to a conventional method, except that the development of the silver staining method is performed with the above-described reducing agent.

As the support used in the present invention, a support made of a polymer carrier such as a polyacrylamide gel, an agarose gel, and an agar gel having a network structure is typically mentioned.

As the immobilizing agent for immobilizing the support carrier containing the test substance, known ones can be used, but preferred examples include those containing a lower alcohol having 1 to 4 carbon atoms and an organic acid. Can be Examples of the lower alcohol having 1 to 4 carbon atoms include methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, isobutyl alcohol, secondary butyl alcohol, and tertiary butyl alcohol. Examples of the acid include monobasic acids such as acetic acid and propionic acid, dibasic acids such as succinic acid and tartaric acid, and tribasic acids such as citric acid. Also, if thiourea is added to the fixing agent, it forms a complex with silver ions in the silver stain,
Silver ions can be easily reduced and the development time can be shortened. In this case, the thiourea concentration in the immobilizing agent is
It should be 0.00001W / V% or more, but usually 0.001 ~ 0.1W / V%
Is preferably used. Further, the alcohol concentration in the fixing agent is not particularly limited, but is usually 10 to 60 V / V
% Is preferably used. Furthermore, the concentration of the organic acid is
Although 5 W / V% or more may be used, usually 10 to 30 W / V% is preferably used.

The fixing treatment may be performed only once, for example, with a solution containing thiourea, a lower alcohol and an organic acid.
Method of treating with a second fixing agent consisting of thiourea, lower alcohol and organic acid after treatment with a fixing agent (ii) First treating with a first fixing agent consisting of thiourea, lower alcohol and organic acid and then lower alcohol And (iii) dividing the fixing agent comprising thiourea, lower alcohol and organic acid into two, and performing the same operation in two steps.
It is performed in two stages, such as a method of performing it twice. In these two-stage treatment methods, it is usually desirable that the alcohol concentration in the first fixing agent is higher than the alcohol concentration in the second fixing agent, and the immersion time for the fixing treatment is 10 minutes for the first fixing agent. degree,
About 15 minutes for the second fixation is enough.

In addition, known sensitizers can be used as the sensitizer used in the pretreatment. Preferred examples include those containing dithiothreitol and glutaraldehyde.

The concentration of dithiothreitol (DTT) in the sensitizer is not particularly limited, but usually 0.0002 to 0.002 W / V% is preferably used. Glutaraldehyde concentration is 0.005W / V
% Or more, and usually 0.005 to 0.2 W / V% is preferably used.

When thiourea is added to the sensitizer, sulfur sensitization of the sensitizer enhances the blackening of silver and makes the contrast of the dyed image clear. The concentration of thiourea to be mixed for such a purpose may be 0.00001 to 0.01 W / V%, but usually 0.0002 to 0.0005 W / V% is preferably used.

The pretreatment is performed by immersing the support immobilized with the immobilizing agent in the sensitizing solution, and the immersion time of about 10 minutes is sufficient.

Furthermore, the silver staining solution used in the method of the present invention is not particularly limited, and known ones can be used. For example, silver nitrate, R-NH ₂ (where R-NH ₂ is ammonia or a primary amine) It is preferable to use a compound containing the compound represented by the formula (1) and caustic alkali in a molecular weight ratio of 1: 4.5 to 9.5: 1.0 to 26.0. The concentration of silver nitrate is not particularly limited, but is preferably 0.05 to 0.4 W / V%.

The silver staining treatment is performed by immersing the fixed and sensitized support carrier in a silver staining solution, and the immersion time is
10-15 minutes is enough.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Staining of serum protein fraction I. Electrophoresis [Preparation of reagent] Supporting carrier Commercially available polyacrylamide gradient gel for electrophoresis SDS-PAG plate 4/20, 1010 (manufactured by Daiichi Kagaku Co., Ltd.) )It was used.

Sample treatment solution containing 4.3 W / V% sodium dodecyl sulfate, 0.02 W / V% bromphenol blue and 30 V / V% glycerol, 0.1
A 25 M Tris-HCl buffer (pH 6.8) was prepared.

Sample Normal human serum diluted 50 times with water is further added with water for 2, 4,
Eight, 16, 32, 64, 128, 256, and 512-fold dilutions were mixed with equal amounts of the sample treatment liquid, and the mixture was boiled in boiling water for 5 minutes to prepare a mixture.

Electrophoresis Buffer A 0.025 M Tris-0.192 M glycine buffer (pH 8.4) containing 0.1 W / V% sodium dodecyl sulfate was prepared.

(Electrophoresis operation)

After the support carrier was attached to the electrophoresis tank, the electrophoresis buffer was filled. Each 5 μm of the boiled sample was applied to a sample well of a support carrier, and electrophoresed at a constant current of 60 mA for about 1 hour.

II. Silver staining [Preparation of reagent] First fixing agent A solution containing 50 V / V% of methanol and 10 W / V% of acetic acid was prepared.

Second immobilizing agent 30V / V% methanol, 10W / V% acetic acid and 0.0025 thiourea
A solution containing W / V% was prepared.

Sensitizer Methanol 50V / V%, Dithiothreitol 0.0005W / V%
And a solution containing glutaraldehyde and 0.1 W / V%.

Silver staining solution A solution containing 0.2 W / V% of silver nitrate, 0.14 W / V% of ammonia and 0.2 W / V% of sodium hydroxide was prepared.

A solution containing 0.02 W / V% of formaldehyde, 0.005 W / V% of citric acid and 0.00005 W / V% of sodium thiosulfate was prepared.

 Terminator A solution containing 10 W / V% citric acid was prepared.

(Staining operation)

The support was immersed in the first immobilizing agent with shaking for 10 minutes, and then immersed in the second immobilizing agent with shaking for 15 minutes. The support was shake-immersed in a sensitizer for 10 minutes, and then washed with ion-exchanged water for 5 minutes. The support was shake-immersed in a silver staining solution for 15 minutes, and then washed with ion-exchanged water three times for 2 minutes each. The support was shake-immersed with a reducing agent until a stained image appeared (about 5 minutes), and then a terminator was added to stop the development.

 The total time required for the above dyeing operation was 66 minutes.

Example 2 Staining of serum protein fraction [Preparation of reagent] First fixing agent Same as in Example 1 Second fixing agent A solution containing methanol 30 V / V% and acetic acid 10 W / V% was prepared.

Sensitizer Methanol 50V / V%, Dithiothreitol 0.0005W / V
%, Glutaraldehyde 0.1W / V% and thiourea 0.00025W
A solution containing / V% was prepared.

Silver staining solution Same as in Example 1 Reducing agent Same as in Example 1 Terminator Same as in Example 1 [Staining operation] Same as in Example 1 Example 3 Staining of serum protein fraction [Preparation of reagent] First fixing agent Same as Example 1 Second fixing agent Same as Example 1 Sensitizer Same as Example 2 Silver staining solution Same as Example 1 Reducing agent Same as Example 1 Terminator Same as Example 1 [Staining operation] Same as Example 1 Example 4 Staining of serum protein fraction [Preparation of reagent] First fixing agent Methanol 50V / V%, acetic acid 10W / V% and thiourea 0.0025
A solution containing W / V% was prepared.

 Second fixing agent Same as in Example 2 Sensitizer Same as in Example 1 Silver staining solution Same as in Example 1 Reducing agent Same as in Example 1 Terminator Same as in Example 1 [Staining operation] Same as in Example 1 Immersion After this (5 minutes), a terminator was added to stop the development.

 The total time required for the above dyeing operation was 50 minutes.

Example 5 Staining of Serum Protein Fraction [Preparation of Reagents] Fixative Methanol 30 V / V%, acetic acid 10 W / V% and thiourea 0.0025
A solution containing W / V% was prepared.

Sensitizer Same as in Example 2 Silver staining solution Same as in Example 1 Reducing agent Same as in Example 1 Terminator Same as in Example 1 [Staining operation] After shaking and dipping the support carrier with a fixing agent for 10 minutes, The plate was immersed in a sensitizer for 10 minutes while shaking, and then washed with ion exchanged water for 5 minutes. The support was shake-immersed in a silver staining solution for 15 minutes, and then washed twice with ion-exchanged water for 2 minutes each. After the supporting carrier was immersed in the reducing agent with shaking until a silver image appeared (5 minutes), a terminator was added to stop the development.

 The total time required for the above dyeing operation was 50 minutes.

Example 6 Staining of Serum Protein Fraction [Preparation of Reagent] A solution containing 30 V / V% of methanol and 10 W / V% of acetic acid was prepared.

Sensitizer A solution containing 50 V / V% methanol, 0.05 W / V% glutaraldehyde and 0.00025 W / V% thiourea was prepared.

Silver staining solution Same as in Example 1 Reducing agent Same as in Example 1 Terminator Same as in Example 1 [Staining operation] Same as in Example 5 Example 7 Staining of serum protein fraction [Preparation of reagents] Fixing agent Example Same as 6 Sensitizer Methanol 50V / V%, Dithiothreitol 0.0005W / V%
And a solution containing 0.00025 W / V% of thiourea.

Silver staining solution Same as in Example 1 Reducing agent Same as in Example 1 Terminating agent Same as in Example 1 [Staining operation] Same as in Example 5 Example 8 Staining of serum protein fraction [Preparation of reagent] First fixing agent Same as Example 1 Second fixing agent Same as Example 2 Sensitizer Same as Example 1 Silver staining solution Same as Example 1 Reducing agent Same as Example 1 Terminator Same as Example 1 [Staining operation] Same as Example 1. [Effects of the Invention] As described above, the present invention provides an improved technique of a silver staining method used for detecting a biological component fractionated on a support. By the method of the present invention, the staining ability was remarkably improved, and the contrast of the obtained stained image was sufficiently enhanced.

Claims (5)

(57) [Claims] In a silver staining method, a support carrier containing a substance to be detected is immobilized with an immobilizing agent, pretreated with a sensitizer, then treated with a silver staining solution, and then developed with a reducing agent. A silver staining method, characterized in that a thiosulfate is contained in the agent. 2. The silver staining method according to claim 1, wherein the thiosulfate is sodium thiosulfate. 3. The silver staining method according to claim 1, wherein the reducing agent further contains formaldehyde and citric acid. 4. A fixing agent containing (1) a lower alcohol having 1 to 4 carbon atoms and an organic acid; (2) a sensitizer containing dithiothreitol and glutaraldehyde; (3) silver nitrate, ammonia or A silver staining kit containing a silver staining solution containing a primary amine and caustic alkali, and (4) a reducing agent containing thiosulfate. 5. The kit according to claim 4, wherein the reducing agent (4) further comprises formaldehyde and citric acid.