STABILIZATION OF DEVELOPED ELECTROPHOREGRAMS

Abstract: A method for determining macromolecules in polyacryl­ amide gels comprising the steps of forming a latent stain image by nucleating the macromolecules in the gel with a palladium tetraamine salt and developing the latent stain image by treating the gel with a physical developing solution comprising dimethylamine borane and a transition metal salt, a tetrazolium salt or a triazolium salt, characterized by the step of stabilizing the developed latent stain image by contacting the gel with a 1-phenyl-2-tetrazoline-5-thione or a salt of 1-phenyl-1H-tetrazole-5-thiol.

Description: The present invention relates to an improved method for determining macromolecules such as proteins, especially when fixed in polyacrylamide gels. The detection of macromolecules such as proteins and polypeptides is extremely important in many areas of biology and clinical medicine such as genetic screening and the diagnosis of genetic diseases. The primary laboratory detection and characterization technique for macromolecules is electrophoresis. Electrophoresis is the movement of charged particles in a matrix under the influence of an electric field. Continuing developments in two-dimensional gel electrophoresis have provided the capability of resolving thousands of macromolecules from complex biological mixtures. US-A-4,552,848 of Yudelson and Johnson discloses a macromolecule electrophoregram visualization procedure of high sensitivity comprising the steps of forming a latent stain image by nucleating polyacrylamide gels containing the macromolecules with a palladium tetraamine salt and developing the latent stain image by treating the gel with a specific physical developer solution. Although this method is useful and has enjoyed widespread acceptance, the resulting developed electrophoregram is somewhat unstable (it is readily oxidized in air by the presence of moisture resulting in image loss). Reducing agents such as sodium hypophosphite have been used as image stabilizers. Such stabilizing solutions, however, require a high concentration of stabilizer compound to effect stabilization. Moreover, these stabilizing solutions fail after the supply of reducing agent is depleted by reaction with oxygen. Example 2 of US-A-4,552,848 discloses a developer having therein 1-phenyl-2-tetrazoline-5-thione at a concentration of about 0.0001%. This reference, however, does not teach or suggest the unexpected image stability resulting from the practice of this invention. Thus, the problems solved by this invention include how to provide improved electrophoregram stabilizer solutions having reduced concentration of stabilizer compound and electrophoregrams having improved image stability. These problems are solved by providing a method for visualizing macromolecules in electrophoregrams comprising the steps of forming a latent stain image by nucleating the macromolecules in the polyacrylamide gel with a palladium tetraamine salt and developing the latent stain image with a physical developer solution comprising dimethylamine borane; and a transition metal salt, a tetrazolium salt or a triazolium salt, and rinsing off the developer solution, the method characterized by subsequently stabilizing the developed latent stain image by contacting the gel with a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-1H-tetrazole-5-thiol. An electrophoregram that consists of a polyacrylamide slab is visualized by nucleation with a palladium tetraamine salt, developed in a physical developer comprising dimethylamine borane, a member selected from the group consisting of a transition metal salt, a tetrazolium salt and a triazolium salt, and optionally an antifoggant, and stabilized by contacting the developed image with a salt of a 1-phenyl-1H-tetrazole-5-thiol or a 1-phenyl-2-tetrazoline-5-thione. Kits comprising a developer containing a palladium tetraamine salt; dimethylamine borane; an antifoggant; and a transition metal salt, a tetrazolium salt or a triazolium salt, characterized in that the kit comprises as a separate package a stabilizer consisting of a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-2-tetrazoline-5-thione, are useful for developing electrophoregrams in accordance with the practice of this invention. These kits can find utility in clinical and laboratory examination of blood and other macromolecules separable by electrophoresis, diagnosis of fetal abnormalities by examination of amniotic fluid, diagnosis of central nervous system disease by analysis of macromolecule patterns in spinal fluid and other applications in which the detection and/or quantitation of macromolecules is desired. The electrophoregram images stabilized by the method of this invention are extremely stable and can be stored for weeks or longer without significant image loss. Further, the concentration of stabilizer required to effect image stabilization is extremely low. The macromolecules to be detected in this invention are preferably proteins, although other macromolecules such as nucleic acids and polypeptides can be detected by the present improved method. The electrophoretic separation using polyacrylamide gel slabs is carried out using any of the procedures well known in the art. Such procedures are described in US-A-4,552,848 noted above. In the preferred embodiments of this invention, the stabilizing compound which is a 1-phenyl-2-tetrazoline-5-thione or a 1-phenyl-1H-tetrazole-5-thiol salt is present in solution at 0.005 to 0.5% by weight of the solvent. Useful herein are the unsubstituted forms of this type of compound: 1-phenyl-2-tetrazoline-5-thione 1-phenyl-1H-tetrazole-5-thiol wherein: X is a basic group such as an alkali metal or an amine, and substituted forms of these compounds, for example, such compounds having the phenyl group substituted with groups such as lower alkyl, having from 1 to 6 carbon atoms, such as methyl, ethyl or isopropyl or other solubilizing groups, provided that the solubility of the substituted stabilizing compound remains sufficient to provide for a 0.005-0.5% solution and that good stabilizing characteristics are retained. The solvent is one in which the stabilizer is soluble, preferably water. Mixtures of solvents may also be used to advantage. For example, the solubility of the stabilizer in aqueous solution may be enhanced by the addition of methanol. Other useful solvents in addition to water include methyl alcohol, ethyl alcohol and acetonitrile. The preferred 1-phenyl-1H-tetrazole-5-thiol salt is the sodium salt having been found to possess excellent solubility and stabilizing characteristics. However, other such unsubstituted and substituted salts including alkali metal salts such as potassium salts and amine salts such as cyclohexylamine salts are also useful herein. Electrophoregram visualization includes the step of forming a latent stain image by nucleating the macromolecules in the gel with a palladium tetraamine salt such as palladium tetraamine chloride, palladium tetraamine nitrate or palladium bis(ethylene diamine) chloride. Palladium tetraamine chloride and palladium tetraamine nitrate yield excellent results in that they possess the proper combination of stability and solubility to sensitize the macromolecular area from the electrophoregram. Optionally, sodium dodecyl sulfate (SDS) sensitizer can be combined with the palladium tetraamine salt in one solution or can be present in separate solutions. The palladium tetraamine salt nucleator is generally present in coverages of from 0.009 to 1.9 mg/m². The development of the latent stain image is accomplished by treating the gel with a developer solution comprising dimethylamine borane as the reducing agent. The reducing agent can be present in amounts between 1 mg/0.093 m² and 200 mg/0.093 m². The developer solution also comprises a transition metal salt, a tetrazolium salt or a triazolium salt physical developer. The transition metal salt, tetrazolium salt or triazolium salt can be present in the amounts between 1 mg/0.093m² and 100 mg/0.093 m². A variety of tetrazolium salts may be used in the practice of the present invention. It is understood that the term "tetrazolium salt" throughout the application includes tetrazolium salts, ditetrazolium salts and tetrazolium betaines. Useful tetrazolium salts are described in "The Chemistry of Formazans and Tetrazolium Salts", A. W. Nineham, Chem. Rev ., 55, 355 (1955). The synthesis of tetrazolium salts and the chelation of formazan dyes are also described in the above reference. Tetrazolium salts useful in the present invention include compounds having the general formula: wherein Ar₁, Ar₂ and Ar₃ may be the same or different and represent phenyl groups or phenyl groups substituted with electron withdrawing groups such as nitro, methoxy and the like or electron donating groups such as alkyl; and Ar₃ can also represent a 4,5-dialkyl-2-thiazolyl group containing 1 to 5 carbon atoms in the alkyl group or a 2-benzthiazolyl group; and X- is an anion such as halide, acetate and tetrafluoroborate. Specific examples of tetrazolium salts and methods for their preparation can be found in Canadian Patent No. 860,873. Triazolium salts may also be employed as reducible dye precursors in the practice of this invention. These are colorless compounds that are reduced by the process of the invention to colored azo-amine dyes. Exemplary triazolium salts are disclosed in Research Disclosure , Item No. 12617, Vol. 126, October 1974 at Table IV. Physical developer baths can be made from substantially equal volumes of a solution of a tetrazolium salt and a solution of a reducing agent. The resulting physical developer bath is considered useful if the tetrazolium salt is not spontaneously reduced to formazan dye but is reduced to formazan dye when palladium (O) or other catalyst is added to the bath. By spontaneously, it is meant that the formazan dye is formed essentially instantaneously without the metal nuclei. The physical developers useful in the present invention can be simple solvent solutions of the tetrazolium salt and the reducing agent. The solvent is one in which the tetrazolium salt is soluble and the corresponding formazan dye as well as the palladium nuclei is insoluble. Mixtures of solvents may also be used to advantage. For example, the solubility of a tetrazolium salt in aqueous solution may be enhanced by the addition of methanol. Other useful solvents besides water include methyl alcohol, ethyl alcohol and acetonitrile. The solution can be saturated with the tetrazolium salt but is useful with as little as about 0.1% tetrazolium salt by weight of the solvent. The transition metal salt deposited from the developer bath must itself be autocatalytic; that is, it must act as a catalyst for further deposition of metal from the developer. This is necessary in order that deposition and development will continue after palladium nuclei are enveloped with heavy metal. With respect to the Periodic Table, suitable heavy metals can be selected from Group VIII metals such as nickel, cobalt, iron, palladium and platinum, Group VIB metals such as chromium and Group IB metals such as copper, silver and gold. Almost any heavy metal salt which is a source of the desired heavy metal ions can be employed. Suitable heavy metal salts useful in the invention include heavy metal halides such as cobaltous bromide, cobaltous chloride, cobaltous iodide, ferrous bromide, ferrous chloride, chromium bromide, chromium chloride, chromium iodide, copper chloride, silver bromide, silver chloride, silver iodide, gold chloride, palladium chloride and platimum chloride, heavy metal sulfates such as nickel sulfate, ferrous sulfate, cobaltous sulfate, chromium sulfate, copper sulfate, palladium sulfate and platinum sulfate, heavy metal nitrates such as nickel nitrate, ferrous nitrate, cobaltous nitrate, chromium nitrate and copper nitrate, and heavy metal salts of organic acids such as ferrous acetate, cobaltous acetate, chromium acetate and copper formate. Baths can be formulated based on a single heavy metal or based on mixtures of heavy metals. The developer solution also preferably contains an antifogging agent to reduce the effects of fogging. It has been found that ethyl cysteine HCl, or dimethyl cysteine dihydrochloride in combination with the dimethylamine borane yields excellent discrimination betwen image and background. Other antifoggants such as dimethylaminoethanethiol HCl are also useful herein. In the preferred embodiments of this invention, the physical development bath contains from 0.1% to 10% of the dimethylamine borane, from 0.1% to 10% of the transition metal salt or tetrazolium salt and from 0.01% to 1% of the antifoggant. The physical developer solution can contain additional materials such as complexing agents such as gluconic acid, tartaric acid, citric acid and ethylene diamine tetraacetic acid. It has been found that the use of gluconic acid produces superior results in sensitivity, stability and fog levels. In addition, it is desirable to include SDS sensitizer in the physical developer solution. Thus, amounts of 0.1% to 1% of SDS in the developer solution result in greater sensitivity without excessive fogging. The development is generally carried out in 10 to 30 minutes. After development is completed, the developed electrophoregram is contacted with a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-1H-tetrazole-5-thiol. To prevent image loss for extended periods of time, subsequent to the development stage the developed electrophoregram preferably is immersed and stored in the stabilizer solution. The following examples are presented to illustrate more fully the invention. EXAMPLE 1 Preparation of Developed Electrophoregrams A developer was prepared as follows: A sensitizer Pd(NH₃)₄Cl₂ was prepared as follows: 3.6 g of PdCl₂ was mixed with 350 ml of deionized water and 50 ml of concentrated ammonium hydroxide. After stirring overnight, all of the PdCl₂ had dissolved. This solution was placed under aspirator vacuum for 8 hours and diluted to 500 ml with water to give a 1% solution of Pd(NH₃)₄Cl₂ whose final pH was 8.7. An electrophoregram of ß-Galactosidase in polyacrylamide gel (0.75 mm thick) containing 10% acrylamide and 2.7% methylene bisacrylamide were fixed in CH₃OH-H₂O (1:1) for at least one hour and then sensitized as follows: The electrophoregram was developed in the solution for 20 minutes at 25°C. After this time, the main protein band could be observed at low protein concentrations. Following development, the electrophoregrams of this example were washed for 1 minute in two changes of distilled water and stored under the following two sets of conditions: After a few days storage at room temperature, Sample #1 showed severe image loss whereas the sample stored in the 1-phenyl-2-tetrazoline-5-thione containing stabilizer solution showed no image loss after several weeks aging. EXAMPLE 2 Effect of Stabilization Following development, the electrophoregrams of Example 1 were immersed in the following stabilizing solutions: and dried by standard techniques on a slab gel dryer. The electrophoregram stabilized in sodium hypophosphite turned appreciably darker upon drying whereas the electrophoregram stabilized in the 1-phenyl-1H-tetrazole-5-thiol salt solution evidenced no image loss throughout the drying procedure.

Claims: A method for determining macromolecules in polyacrylamide gels, said method comprising the steps of a) forming a latent stain image by nucleating the macromolecules in the gel with a palladium tetraamine salt; b) developing the latent stain image by treating the gel with a physical developer solution comprising dimethylamine borane; a transition metal salt, a tetrazolium salt or a triazolium salt; and c) rinsing off the developing solution, characterized by subsequently stabilizing the developed latent stain image by contacting the gel with a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-1H-tetrazole-5-thiol. A kit for the determination of macromolecules in polyacrylamide gels comprising a developer containing a palladium tetraamine salt; dimethylamine borane; an antifoggant; and a transition metal salt, a tetrazolium salt or a triazolium salt; characterized in that the kit comprises as a separate package a stabilizer consisting of a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-1H-tetrazole-5-thiol. The method according to claim 1 or the kit according to claim 2 wherein said transition metal is nickel. The method or kit of any previous claim wherein said stabilizer is the sodium or cyclohexylamine salt of 1-phenyl-1H-tetrazole-5-thiol. The method or kit of any previous claim wherein the stabilizer is present in solution at 0.005 to 0.5% by weight of the solvent. The method or kit of any previous claim wherein said palladium salt is palladium tetraamine chloride. The method or kit of any previous claim wherein said palladium salt is in a first solution and the dimethylamine borane is in a second solution.

Inventors: YUDELSON, JOSEPH SAMUEL
Applicants: EASTMAN KODAK COMPANY (A NEW JERSEY CORPORATION)
Application year: 1986
Main classification: G01N33
IPC codes: G01N33, G01N27