JPH0141222B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an ultrasensitive metallic silver staining method for polypeptides fixed on synthetic gels, particularly polyacrylamide gels. The detection and identification of polypeptides is of fundamental importance in many fields of biology and clinical medicine. In some endeavors, such as the genetic selection of mutational events, the monitoring of pathophysiological changes, and the diagnosis of genetic diseases, the efficiency of research is due to the use of polypeptides that can be detected and identified in cellular extracts of body fluids. is directly proportional to the number of Further, polypeptides, hormones, etc. that exist in trace amounts are extremely important for various medical reasons. Electrophoresis (commonly defined as the movement of charged particles in solution under the influence of an electric field) is a major laboratory detection and identification method, especially for polypeptides and other micromolecules, e.g. nucleic acids. It is useful as a material. Electrophoresis can also be used to analyze small particles such as viruses, cells, subcellular
-cellular), cell organelles, and organic molecules such as steroids and amino acids. As a result of continued development of two-dimensional gel electrophoresis,
It has become possible to separate thousands of polypeptides from complex biological mixtures. However, the inability to detect polypeptides present at low concentrations limits the application of this method, particularly in clinical screening for pathological conditions, endocrinology, mammalian metabolism, developmental biology, and immunology. This improved gel electrophoresis method greatly increases the separation of polypeptides, so that visual detection methods using common polypeptide dyes are no longer suitable. The most commonly used general polypeptide dye is Coomassie blue (Coomassie blue).
Blue), which can be considered a prototype. This class of dyes relies primarily on electrostatic attraction between the dye and the polypeptide, stabilized by van der Waals forces. In fact, Coomassie Blue and a variety of other dyes exhibit particular affinities for polypeptides with specific charges.
Coomassie blue, an acidic dye, stains basic polypeptides most strongly, and crystal violet is the most effective dye for acidic polypeptides. Other dyes for which the quantitative properties of staining are already known include Amido Black and Fast Green.
Green) and Fe ²⁺ -bazophenanthroline sulfonate. In contrast to this, Remazol
Brilliant Blue Earl (Remazol
The Brilliant Blue® method relies on covalent bonding between the dye and the polypeptide. For Coomassie Blue, polypeptide concentrations of 0.05 to 2 μg were determined using relative spot area parameters by staining for 30 minutes in a 1.5 mm diameter gel.
It was found that there is linearity in the range of . When staining is carried out for 60 minutes instead of 30 minutes, the slope of the relationship between area and concentration increases, and at 1 μg or more, it reaches saturation and loses linearity. Fluorescent amines can react with the terminal amino and ε-amino groups of polypeptides in gels to obtain a sensitivity at least equal to that of Coomassie Blue, with 1 to 1 per "spot".
Linearity in the 7μg range. MDPF (2-methoxy-2-4-diphenyl-3(2H)-furanone)
When electrophoresis is performed after fluorescently labeling a polypeptide using the method, linearity is obtained in the range of 10 ng to 10 μg of protein. However, when electrophoresis is performed after fluorescently staining a polypeptide, the electrophoresis pattern may change. There are other methods that do not require modification of the polypeptide prior to electrophoresis. Such methods include photodensitometric scanning of absorption at 280 nm, binding of radiolabeled or fluorescent ligands such as concanavalin A to the glycoprotein, binding of antiserum to polypeptides on the gel surface, and There is a method of staining the polypeptide portion of A method for staining specific polypeptide parts is to stain the side chains of carbohydrates with PAS.
sulfhydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid), copper polypeptides with cyanide-tetrazolium, cadmium polypeptides with dipyridyl-ferrous iodide, and Ca ² -polypeptides with ⁴⁵ Ca autoradiation. There is a method of staining with graphie. Radioactive detection methods are more sensitive than staining methods but are generally impractical. In vivo, radioactive labels can alter cellular metabolism, and it is not possible to label human polypeptides. In vitro, radiolabels have the disadvantage of altering the electrophoretic mobility of the polypeptide. Furthermore, radioactive reagents can be very expensive in some cases and give rise to the problem of "autoradiographic diffusion" when exposed for long periods of time to detect trace amounts of polypeptides. Polypeptides labeled with radioactive precursors can be detected by autoradiography or fluorography. These methods are standardized and used quantitatively. Quantitation can be easily performed by placing a set of radiographic standards adjacent to the gel when exposing the film. Fluorography requires the gel to be impregnated with a scintillating fluorophore. Fluorography is most useful when labeling with low energy beta emitters or when increased detection sensitivity is required. In order to use fluorography quantitatively, it is necessary to cover it with a film in advance.
Recently, a method has been announced in which polypeptides are chemically radiolabeled in vitro and then subjected to electrophoresis. Such a method is based on the ε-
There is reductive methylation of the amino group and reductive methylation of the α-amino group of the N-terminal amino acid. This can be done with formaldehyde and sodium cyanoborohydride. C ¹⁴ formaldehyde or ³ H sodium cyanoborohydride can be used as radiolabels. N-succinimidyl [2,3- ³ H]propionate can be used to covalently label the terminal group and the ε-amino group in vivo or in vitro. All of these methods unfortunately alter the mobility of the labeled polypeptide. The above staining methods are difficult to perform, dangerous, time-consuming, and detect proteins present in low or trace concentrations when the polypeptide is not heavily labeled. The sensitivity is not sufficient for this purpose. One problem exists when using body fluids, such as cerebrospinal fluid or amniotic fluid. These body fluids are generally difficult to collect in large quantities and contain large amounts of certain types of proteins. Such amounts of protein change the electrophoretic pattern when a sufficient amount of sample is analyzed to examine specific trace polypeptides. A highly sensitive silver staining method for analyzing polypeptides in polypeptide gels has recently been developed. These methods suffer from the disadvantages of consuming too much silver, being too complex, and although in most cases are more sensitive than silver-free dyes, they are less sensitive or reproducible than the improved method of the present invention. inferior in gender. Mr. Kerenyi and Mr. Gallias, “Highly sensitive methods for detecting proteins by electrophoresis, immunoelectrophoresis, and immunodiffusion agents,” Clinical Chemistry Acta, Vol. 38, pp. 465-6.
467 (1972) discloses a silver dye for proteins in agar gels. In this document, the gel is immersed in potassium ferrocyanide and then in two different developers. The first solution contains sodium carbonate and water, and the second solution contains ammonium nitrate, silver nitrate, tungsten silicate, and formaldehyde. The use of polyacrylamide gels has been mentioned as a possibility. Mr. Kerenyi and Mr. Gallias, “On the problem of quantitative artifacts physically occurring in silver staining in electrophoresis,” Clinical Chemistry Acta, Vol. 47, pp. 425-
436 pages (1973), continued the research on silver staining published in 1972. It has been discussed to prevent artifacts from occurring during dyeing. After soaking the protein-containing gel in potassium ferrocyanide, Mr. Bielhik writes in "Agar gel electrophoresis of unconcentrated cerebrospinal fluid", Journal of Neurology, Vol. 209, pp. 59-63 (1975). A method for silver staining in agar gel using two solutions is disclosed. The first solution is a sodium carbonate solution and the second solution contains ammonium nitrate, water, formaldehyde, tungsten silicate and silver nitrate. Reported results, although generally favorable, are mixed. Mr. Kerenyi and Mr. Gallias (1972) above
The explanation is that although this is a repeat of the method, it does not seem to be generally accepted because a separate band in the gamma region of the electropherogram is not detected and a large number of artifacts are found. is being done. Beerheek himself has found difficulties with bands in several areas. Kulchaer Lowenthal and Van Soom, “Electrophoresis of cerebrospinal fluid proteins without preconcentration,” Acta Neurology Berg, Vol. 79, pp. 335-
337 (1979) discloses a silver staining method using two solutions after soaking a protein-containing gel in potassium ferrocyanide. The first solution contains sodium carbonate and the second solution contains ammonium nitrate, water, formaldehyde, tungsten silicate, and silver nitrate. The article concludes that this staining is comparable to that obtained for conventional electrophoresis using Amidoblack staining for concentrated cerebrospinal fluid. Switzer, Merrill and Shiffrin, “Highly Sensitive Silver Staining for Detecting Proteins and Peptides in Polyacrylamide Gels,” Anatomical Biochemistry;
Silver staining is disclosed in Vol. 98, pp. 231-237 (1979). The method involves soaking the gel in various methanol and acetic acid mixtures for at least 25 hours, soaking the gel in paraformaldehyde solution for 0.5 hours, soaking the gel in cupric nitrate and silver nitrate solutions for at least 0.5 hours, Diamine solution (silver nitrate,
Proteins are fixed by soaking the gel in NaOH (a mixture of NH ₄ OH and ethanol) for 10 minutes and then reducing the gel dye twice with formaldehyde and citric acid. This dye was found to be more than 100 times more sensitive than Coomassie Blue, which is comparable to autoradiography. Merrill, Switzer and Van Keulen et al. ``Trace polypeptides in cell extracts and human body fluids detected by two-dimensional electrophoresis and high-sensitivity silver staining'',
International Academic Society, USA, Vol. 76, pp. 4335-4339
(1979), using the dyes disclosed by Switzer, Merrill and Shiffrin et al. in Anatomical Biochemistry (1979). Several potential clinical uses are discussed, and it is demonstrated that this dye is more sensitive than Coomatsie Blue and is less expensive and faster than autoradiography. Orclay, Kirsch and Morris et al., “A simple ultrasensitive silver stain for detecting proteins in polyacrylamide gels,” Anatomical Biochemistry, Vol. 105.
Volume, pp. 361-363 (1980), the above-mentioned Switzer, Merrill and Shiffrin et al. (1979)
Discloses the application of silver dye, which was first disclosed by (2011). In this disclosed method, (1)
Soak the gel in glutaraldehyde for 30 minutes, (2) rinse and soak the gel in water for at least 2 hours, (3) add ammoniacal silver solution (a mixture of NH ₄ OH, NaOH and AgNO ₃ ), (4 ) The gel is transferred into a mixture of citric acid and formaldehyde and (6) washed in water for at least 1 hour. To simplify the initial operation, cupric nitrate,
The benefits are described as eliminating the silver nitrate step and reducing the amount of silver required. When repeating this method in connection with the present invention, when comparing the dyeing method of Oaklei, Kirsch and Morris above with the improved method of the present invention,
The sensitivity was found to be significantly low (approximately 50%). There is still no patent disclosing silver dyes for proteins or with greater certainty than the above monograph. US Pat. No. 3,873,433 does not disclose a silver dye, but rather a method for staining proteins by forming an organic complex with a divalent element such as calcium or magnesium. US Pat. No. 4,167,467 does not disclose a silver dye, but rather a method for quantifying lipoprotein free cholesterol by enzymatic measurement using a cholesterol oxidase substrate. The present invention provides a silver staining method that can detect polypeptides at 0.01 ng/mm ² . The method is similar to photochemistry and involves (1) fixation of the polypeptide, (2) photographic reversal using an oxidizing agent, (3) formation of a latent image, preferably with silver nitrate, and (4) visible staining. It consists of a continuous process of development and development. The silver staining method of the present invention can be used for clinical and laboratory tests of blood-derived polypeptides and other polypeptides separated by electrophoresis, for prenatal diagnosis of fetal abnormalities by testing amniotic fluid, and for prenatal diagnosis of fetal abnormalities by testing amniotic fluid. diagnosis of central nervous system diseases by analysis of polypeptide patterns; analysis of cellular protein patterns for physiological and pathophysiological studies; applications in visualization and quantification of polypeptide hormones; detection of proteins or polypeptides; There are other applications that require quantitative determination. Any gel composition suitable for use in electrophoresis can be used in the present invention. Typical gels are polyacrylamide, agarose and cellulose acetate. Cellulose acetate and agarose gels give a brownish background when used in the method of the invention (agarose is slightly darker), so polyacrylamide is preferred. All of these gels are suitable if this brownish background can be eliminated. Although the staining method of the present invention has its primary use in staining polypeptides on gels, this staining method (sometimes omitting the fixation step) has not yet been applied to the histology of tissue sections such as biopsies. It is useful in performing complex staining and protein analysis of polypeptides isolated from restricted materials. Next, preferred embodiments of the present invention will be described. The silver staining method of the present invention consists of the following four basic sequential steps: (1) washing and fixing, (2) photographic reversal, and (3)
It consists of latent image formation and (4) development. The methods of the invention can be used in combination with gel electrophoresis, polypeptide assays and tissue staining. Because the silver staining of the present invention is highly sensitive, this method is most useful in combination with electrophoresis, especially two-dimensional electrophoresis. The method of the invention is described below, particularly in relation to electrophoresis using polyacrylamide gels. First step (washing and fixing). After introducing a sample that is thought to contain a polypeptide into a gel using a conventional method, it is necessary to elute from the gel substances that may fix the polypeptide in the gel and interfere with the staining operation. Examples of substances to be eluted include ampholytes used in the isoelectric concentration step of two-dimensional electrophoresis, reducing substances in the electrophoresis slow solution,
There are detergents (eg sodium lauryl nitrate) used in electrophoresis. Washing and fixing steps are common in all polypeptide gel staining methods, but are particularly important when small amounts of polypeptide are present. Common fixing agents such as glutaraldehyde, oxides of heavy metals such as mercury, lead and osmium, formaldehyde, paraformaldehyde, trichloroacetic acid and acetic acid can be used. However, glutaraldehyde and heavy metal oxides pose health risks and should not be used. Trichloroacetic acid (10%), acetic acid, formaldehyde or paraformaldehyde are preferred fixatives, but these all usually have to be washed from the gel with methanol or ethanol before staining. The most preferred fixing agent is ethanol and/or methanol (approximately
(10-50%) and acetic acid (about 5-20%), with about 10% ethanol and about 15% acetic acid being optimal. The effect of fixatives is not precisely understood.
The above fixatives have been used in histology and protein electrophoresis for many years. It has at least been shown that the relative concentration of protein is kept constant using the fixative described above, without using modern assay methods using external and internal standard proteins for this gel. Typically, the gel is fixed by soaking in the fixative for about 5 to 15 minutes and then repeatedly soaked in fresh fixative for up to three times. If a fixing agent other than an acetic acid-methanol and/or ethanol mixture is used, rinsing with methanol and/or ethanol is preferably followed. No rinsing is necessary when using the most preferred fixing agents, acetic acid-methanol and/or ethanol mixtures. Second step (photo reversal). After fixation, the polypeptide is treated with an oxidizing agent to perform photographic reversal and prevent silver staining of non-polypeptides. Suitable reagents include dichromate, potassium permanganate, molecular oxygen, potassium ferricyanide, iodine and quinones. Among these, dichromic acid, potassium dichromate and molecular oxygen are preferred. Potassium dichromate is particularly preferred. Potassium ferricyanide was initially tried in the present invention but was found to have poor sensitivity. In the experiment, 0.03M potassium dichromate gave the best results. Purified dichromate fluid is known for histological staining (Lillier and Fuhrmann, Histopathological Techniques and Practical Histochemistry, 4th edition, Matsuku Grau).
Hill Book Company, New York;
(1976), p. 54), the fundamental principle of its use is quite different from that of the present invention. Typically, this step is carried out by soaking the fixed polypeptide gel for about 3 to 10 minutes (preferably about 5 minutes) in an aqueous solution, preferably containing potassium dichromate and a small amount of nitric acid. Most preferred is a solution of about 0.03M potassium dichromate and about 0.0015-0.03 (preferably about 0.0016)M nitric acid. In a generally less preferred embodiment, this step is followed by a quick rinse of the gel with deionized water. Third step (latent image formation). At this point, the polypeptide gel is immersed in a photosensitive salt solution and, if necessary, activating irradiation is performed. Although silver salts, gold salts, platinum salts, palladium salts, and/or iridium salts can be used, silver salts are preferred because they are relatively easily available and inexpensive. Silver nitrate is the most preferred salt. The concentration of this salt used is inversely (but not inversely) related to gel thickness. Therefore, in the case of an extremely thin gel of 0.01 mm or less, the concentration of the metal salt used must be higher, but as the gel becomes thicker, a thinner concentration is required. Typically 0.8mm
It was experimentally found that a 0.01M silver nitrate aqueous solution was the most effective when using a polyacrylamide gel with a thickness of . Using an appropriate concentration of oxidizing agent (eg dichromate) in step 2 above eliminates the need for light or other irradiation in step 3. In fact, light is undesirable because it magnifies the background. It is therefore desirable to prepare the dye in the dark or under a red safety light such as those used in photographic darkrooms. This operation is similar to photochemical reversal in photography, where photographic reversal is performed with a suitable oxidizing agent without exposure to light. By analogy, most commercial photographic reversal operations (such as those in the manufacture of color slide film) are entirely chemical processes and do not utilize actuation by light or other illumination. If the gel thickness is approximately 1 mm or more or DNA
It is possible, and even preferred, to use photoirradiation under certain conditions, such as dyeing certain organic polymers such as. This irradiation is carried out by conventional methods, but must be sufficient to effect the photochemical reaction. Light irradiation from infrared and/or thermal radiation has been found to be effective. If this irradiation is used, it is preferably carried out during the first few minutes of immersion in the photosensitive salt to provide maximum sensitivity. A bright uniform light source can be used, such as a 160 watt fluorescent grid lamp, which typically produces light equivalent to a 1500 watt tungsten source. When using a saturated light source of this intensity, a 5 minute exposure was found to be appropriate. As an example, when polypeptide gels are exposed to ambient light and heat in the laboratory without a light source of this intensity, the sensitivity of this staining method (expressed in terms of spot concentration of polypeptide) decreases by more than 50%. did. The polypeptide gel should be soaked in the silver nitrate solution for about 10-30 minutes, preferably about 20 minutes. When a light source with sufficient intensity is used, light irradiation may be performed for only a fraction of the immersion time, but the immersion time and irradiation time for silver nitrate may be appropriately determined so that they are performed equally and simultaneously. Fourth step (development). Next, this polypeptide gel is removed from the photosensitive salt solution and rinsed if necessary.
It is then treated with a reducing agent or developer. This photosensitive salt solution can be recycled by measuring it and adjusting it to the desired concentration. A large number of developers known in the photographic art can be used. These developers include metal compounds of iron, tungsten, vanadium and molybdenum and organic compounds including hydroquinone, pyrogallol, p-aminophenol, p-phenylenediamine, paraformaldehyde or formaldehyde. Among these, organic compounds are preferred, and formaldehyde and/or paraformaldehyde are most preferred. When formaldehyde and/or paraformaldehyde is used, it is preferably used as a mixture with an alkalizing agent. The alkalizing agent is typically sodium carbonate or sodium metaborate. It is desirable to immerse the polypeptide gel in the reducing agent so that the development of the latent image continues until the polypeptide gel contacts the reducing agent, at which point the polypeptide gel is removed from the photosensitive salt. This prevents the loss of photosensitivity-derived metals in the gel, which would lead to a decrease in sensitivity. In particular, the protein gel should be soaked in the reducing agent for about 10-20 minutes immediately after removal from the photosensitive salt solution. When using formaldehyde and/or paraformaldehyde, a typical reducing solution is an aqueous mixture of 0.5 ml of formaldehyde and 0.3 M sodium carbonate per liter. Immediately after removing the polypeptide gel from the first portion of the reducing solution, it is desirable to then immerse the polypeptide gel in a second portion of the same solution for about 10 to 20 minutes with gentle agitation. This latter immersion time can vary and should generally be about 15 minutes until the desired intensity of staining is achieved.
Once staining is complete, discard the reducing (developer) solution and thoroughly wash the polypeptide gel with distilled water. If necessary, the polypeptide gel is preferably immersed in an acid solution immediately before being removed from the reducing solution and washed. This acid solution is an aqueous solution of acetic acid, citric acid or hydrochloric acid, preferably acetic acid (1-3%). This acid solution has the function of stopping the development of the dye and should be used for about 5 minutes. Continuing tests using the photochemical silver staining of the present invention revealed that the sensitivity to trace amounts of protein is mainly due to the diffusion of silver from the latent image and the gel during the development process. This can be overcome by recycling the staining operation by the following embodiment. After immersing the gel in an acid solution (repeating immersion in the acid solution and washing one or more times if necessary) and stopping development, the third step (latent image formation) and fourth step (development) are repeated. This circulation from the photosensitive salt step onwards reduces the consumption of silver caused by diffusion of silver from the gel during development. When a 1 mm thick gel is used, the concentration of silver deposited in each band or spot reaches its maximum in about 20 minutes in the sodium carbonate and formaldehyde solution. Addition of fresh sodium carbonate and formaldehyde will not increase the concentration of spots at this point. However, by recycling (by replacing silver nitrate) additional concentrations can be achieved. If the polypeptide requires further staining, one or more cycles can be performed; however, repeated cycles may cause the background to become darker. Application examples of the present invention include the following. (1) Routine test for polypeptides in blood separated by gel electrophoresis. Due to its sensitivity and linear relationship with polypeptide concentration, the staining method of the present invention can be used to analyze polypeptide abnormalities or pathologies (such as occur in liver disease and heart disease). (2) Prenatal diagnosis of fetal abnormalities by detecting abnormal polypeptide patterns in amniotic fluid that are associated with genetic abnormalities and developmental abnormalities. (3) Diagnosis of central nervous system diseases by analyzing polypeptide patterns in spinal fluid. Before silver staining for the polypeptides of the present invention was developed, approximately 40
Various polypeptide bands were observed. It is now possible to identify over 400 polypeptides using the staining method of the invention. (4) Detection of polypeptide hormones and quantification of their concentrations in serum, spinal fluid, urine, etc., as appropriate. (5) Analysis of cellular polypeptide patterns for genetic, physiological, and/or pathological research. (6) Purification of polypeptides by identifying contaminants at lower concentrations than previously possible. (7) Detection of polypeptides present at low concentrations in urine and spinal fluid without high concentrations. (8) The staining method of the present invention can be used in combination with a high-resolution polypeptide mapping method to easily quantify metabolic phenomena, evaluate pathophysiological processes, and detect mutations. (9) Evaluation of pharmacotherapeutic effects on metabolic pathways monitored by polypeptide concentrations. (10) Diagnosis of tumors by observing characteristic polypeptides associated with malignant cells. (11) In general, the staining method of the present invention can be used in combination with analytical techniques, especially when the polypeptide content of the evaluation sample is low. (12) Polypeptide (protein) assay by precipitating the polypeptide on paper (particularly cellulose nitrate paper) and staining it by the method of the present invention. Fixation is not required, but the staining procedure is otherwise the same. (13) Tissue staining can be performed using the method of the present invention. Staining of tissue sections, such as those obtainable by biopsies, is particularly sensitive when performed according to the method of the present invention. (14) Chromosomes can be stained using the method of the present invention. It is possible to discern patterns of bands with even greater sensitivity, and perhaps even more bands can be shown. This is particularly useful in prenatal diagnosis and positive as well as relative identification of specific cells. Examples Example 1 (Silver staining of the present invention) Mr. O.Fouraire, Anatomical Biochemistry, Vol. 250,
Polypeptides were separated by the two-dimensional electrophoresis method disclosed on pages 4007 to 4021 (1975). Ten
% acrylamide second direction gel was 16 x 12 cm and 0.8 mm thick. anchors the polypeptide,
Excess sodium dilauryl nitrate was removed from the gel by three 10 minute rinses of 200 ml with an aqueous solution of ethanol (10%) and acetic acid (5%). The polypeptide gel was then immersed in a 200 ml solution of 0.034 M potassium dichromate and 0.032 N nitric acid for 5 minutes. The gel was washed three times for 2 minutes each with 200 ml of deionized water and then dissolved in 200 ml of 0.012 M silver nitrate.
Soaked for 20 minutes. This was then quickly rinsed with two 300 ml aliquots of a developer containing 0.21 M sodium carbonate and 0.5 ml of commercially available formaldehyde in one part. The gel was gently stirred in a third aliquot of the solution until the image reached the desired concentration. The developer was discarded and 100 ml of 0.2M acetic acid was added to stop the development. The gel was washed twice with 200 ml of water, then placed in a plastic bag and sealed. Maximum sensitivity is obtained by exposing the gel to relatively intense, uniform light or heat for the first few minutes in silver nitrate. A uniform intensity fluorescent light source gave the best results. It was found that when the thickness of the gel exceeded 1 mm, the sensitivity was lost. The silver stain of the present invention was found to be at least as sensitive, and perhaps even more sensitive, than the histologically derived silver stain (FIG. 1). The concentrations obtained using these two dyes were proportional to protein concentration over a wide range. The number of spots of polypeptide produced on two-dimensional gels demonstrated that photochemical silver staining was at least as effective as histological staining in detecting the presence of polypeptide in gels. The two dyes showed similar specificity for polypeptides. When E. coli or human cell lysate was treated with protease K (a polypeptide-degrading enzyme), all isolated spots on the gel disappeared. The usefulness of this dye is illustrated in FIGS. 2 and 3. Figures 2 and 3 show electrophoretic patterns of cerebrospinal fluid (CSF) and plasma from various parts of the avian species. CSF was obtained from the lateral ventricle, Cisterna magna and lumbar space of six Rhesus macaques. albumin,
quantified the distribution of numerous polypeptides including α1-antitribusin, haptoglobin, immunoglobulin G (light and heavy chains, Gc globulin, α2HS glycoprotein, α1-antichymotrypsin, α1-β-glycoprotein, and transferrin). These were found to be present at lower concentrations in CSF from the lateral ventricles. Certain CSF polypeptides, which are not found in plasma, were also found to be present at lower concentrations in lateral ventricular CSF. However, other CSF polypeptides, some of which are indicated by arrows in the figure, were not observed in the lateral ventricular CSF.This suggests that changes in individual polypeptides may occur in subregions of the CSF. Hill, Matsukukenil, Matsukugutskin, Coldstein, and Sabieux et al., Mayo Clinical Journal,
In Volume 33, pp. 686-698 (1958),
One-dimensional electrophoresis was used to demonstrate gradients in cerebrospinal fluid for albumin, prealbumin, and four globulin fractions. These investigators found that the concentrations of albumin and the four globulin fractions were lower in the lateral ventricular CSF, and that the lateral ventricular CSF had a relatively higher concentration of prealbumin. Using immunological methods, we discovered nine antigenic species in CSF that were not present in serum. Recently, a method for preliminary separation of CSF-specific proteins by affinity chromatography and isoelectric focusing has been proposed. The silver-stained two-dimensional electrophoresis gel of the present invention enables the quantification of more than 300 types of polypeptides in CSF and the direct visual observation of protein fluctuations in CSF and other tissue fluids. FIG. 1 above compares the primitive histochemically derived stain (Switzer, Merrill and Shiffrin (1979)) with the improved silver stain of Example 1 of the present invention. This is Escherichia coli (E.
coli) lysate gel pattern versus concentration of all polypeptide spots within a small area of the lysate gel pattern. The slope was 1.08, the Y-intercept was -8.1, and the correlation coefficient was 0.94. The gel was placed adjacent to the National Bureau of Standards, graduated with photographic density standards, and
Photographed with X120mm film (Kodatsuk). This photographic image was then measured using a 1000HS scanning densitometer (Obtronix International Inc., Kelmsford, Mass, USA).
The images were scanned with a resolution of 100 microns. Image densities were converted to optical density units using calibration density standards. This transformation normalized the gel image for variations important in photography and scanning densitometry. Measurements were performed using an IP5000 image processor (De Anza Systems, San Francisco, Calif.) and a PDP11/60 computer (Digital Equipment, Marlboro, Mass., USA) with background subtraction and using the same measurement window. . The first gel pattern is
10 μg of E. coli lysate protein was prepared by subjecting it to two-dimensional gel electrophoresis according to the method of Oferlele described above. FIG. 2 shows a two-dimensional electrophoretic pattern of spinal fluid and plasma of a bird of prey stained with the silver dye of Example 1 of the present invention. Samples were collected from each adult rhesus macaque by cannulating the loin or cavity, cannulating the lateral ventricle, or inserting a vein into the vein. CSF samples at 4°C
Dialyzed against 10% polyethylene glycol
Concentrated twice. 15 of each concentrated CF sample
A microliter and one microliter of plasma were subjected to electrophoresis. In the figure, the arrow on the lateral ventricular CSF pattern indicates a relatively large amount of concentrated polypeptide in the lateral ventricular CSF. FIG. 3 is an enlarged composite view of a small area of the gel shown in FIG. This is a region where large differences in polypeptide patterns are observed.
The arrows in the figure indicate polypeptides that are relatively concentrated in lateral ventricular CSF. Example 2 (General Sample Preparation and Electrophoresis) Sample preparation: Human peripheral blood lymphocytes were incubated with a Ficoll-Hypaque gradient obtained by needle-pricking a vein under sterile conditions in a heparinized tube.
Lymphocytes were separated by centrifugation at 450×G for 40 minutes.
The cells were washed twice with phosphate buffered saline (PH7.4) and frozen-thawed three times. 2% sodium dilauryl sulfate 5% mercaptoethanol, 20%
Glycerol, 2% Biolite 3/10 (Biorad, Richmond, California, USA)
and 2% Nonidet) P-40 (BRL,
Bethesda, MD, USA) containing lysis solution (100 microliters/sample) was added and the sample was heated at 95°C.
and heated for 5 minutes. After heating, the sample was quickly cooled to room temperature in an ice bath and centrifuged at 12,000 G for 2 minutes. The supernatant was stored at -70°C. Plasma samples were prepared by heating heparin-treated venous blood with 6 times the volume of lysis solution to denature proteins and stored at -70°C. Two-dimensional electrophoresis of lymphoid cells and Escherichia coli proteins was carried out according to the electrophoresis procedure. Isoelectric focusing was performed at 500 volts for 20 hours using a mixture of 1.6% (5-7) and 0.4% (3-10) ampholines. Second direction electrophoresis was performed using a 10% homogeneous acrylamide gel (0.8 mm thick) and a 4.75% acrylamide stacking gel at 20 mA/gel. Plasma protein standards are prepared by mixing 5 microliters of denatured plasma with 10 microliters of 20% glycerol and adding this solution to 0.1% glycerol in a trough.
It was prepared by placing it on the surface of a PAGE gel in the presence of SDS. After electrophoresis at 20 mA, the gel was sliced parallel to the current direction, cut into long strips, and diluted with methanol (50%) and acetic acid (12
%) and stored for use. Purified denatured low molecular weight and high molecular weight marker proteins were placed in preformed wells and subjected to polyacrylamide gel electrophoresis in the same manner as plasma proteins. Staining, a common histochemically derived stain as described by Switzer, Merrill and Shiffrin et al. (1979), was performed and compared with the recently developed silver staining method of the present invention. Histochemical staining is performed in "Brine Behavioral Evaluation", Volume 2, Page 213~
It is based on Mr. De Olmos' copper-silver method described on page 237 (1969). This requires 915 g of silver per gel for 3 hours. Example 3 (Silver staining of the present invention) (1) A gel injected with a polypeptide sample was fixed with a solution of 10% methanol and 5% acetic acid, and then washed twice with the same solution for 10 minutes each. The remaining sodium dilauryl sulfate was removed. (2) Next, add this gel to 0.03M potassium dichromate.
Immersed in 200ml of solution containing 0.0016M nitric acid for 5 minutes,
Then add 200 ml of deionized water for 2 minutes each.
Turn it. (3) Next, while illuminating this gel with a uniform light source,
Soaked in 0.012M silver nitrate (200ml) for minutes. (4) Discard the silver nitrate and aliquot the gel into 300 aliquots of a developer containing an aqueous solution containing 0.3 M sodium carbonate and 0.5 ml of commercially available formaldehyde in 1 part.
ml twice immediately. The gel was then gently stirred in a third aliquot of developer until the image reached the desired density (approximately 15 minutes). After staining was completed, the developer was discarded and the gel was thoroughly washed with distilled water or placed in a 0.02M acetic acid stop solution for 5 minutes and then washed with distilled water. To measure concentration, place a graded concentration standard (to facilitate standardization of gel concentration) beside this gel.
I took the photo with 120mm Tri-X film (Kodatsuk). Negatives were scanned with a P-1000HS scanning densitometer (Optronics International Inc., Kelmsford, Mass) at 100 micron resolution using the "3D" optical density range. This data was processed using a DeAnza 5000 image processor.
Processed by PDP11/60 computer. The same gel solution was measured for specific polypeptide bands and spots. Density standards on the negative were used to standardize the computer images and correct for variations in photographic and manipulation techniques. Background was assessed by subtracting low concentration values near the polypeptide or, in some experiments, by creating a concentration histogram of a subregion of the gel and identifying the modal concentration. To measure the size of polypeptide bands, the area of bands with a concentration greater than the background concentration (as determined by the concentration histogram) was calculated. The light effect was investigated using plasma gel strips. The light source used for this experiment and all staining was
A 160 watt fluorescent grid lamp (Aristo Grid Lamp Products, Inc., Washington, NY, USA), which emits light equivalent to a 1500 watt tungsten source. Analysis of Examples 2, 3 and Other Experimental Results Staining of polypeptides by the silver staining method of the present invention is actually a process that is significantly influenced by three variables: exposure, polypeptide concentration, and development time. be. Omitting the intense light exposure during the silver nitrate step resulted in a concentration of polypeptide spots that was less than 50% of the concentration in the same gel obtained with intense light exposure. Exposure by light source when using saturated exposure 5
It was found that no increase in sensitivity occurred after minutes. The gel could be stained with different polypeptide concentrations such that their concentrations were close to each other as shown in FIG. Analysis of the overall concentration (background subtracted) of each of the 45 polypeptides on this gel revealed that the concentrations were proportional between gels, as long as the polypeptide concentrations did not differ by more than 10 times. . The relationship between polypeptide concentration and density is similar to the relationship between total exposure and density found in photography. Many polypeptides showed saturation of staining when excess amounts of the polypeptide mixture were applied. This is just as the area of a photographic negative exhibits saturation under overexposure conditions. This saturation of staining caused the correlation coefficient of the concentration versus concentration plot to become smaller. The lower the amount of polypeptide mixture applied, the more polypeptides will fall on the linear portion of the polypeptide staining curve. Analysis of stained purified polypeptides shows that the concentration increases proportionally upon development and that the staining
It can be seen that when using a 0.8 mm thick gel, the sensitivity is linear from the limit (0.1 ng/mm ² ) to approximately 2 ng/mm ² . By photographing the gel during development and then measuring the concentration, a series of polypeptide concentration vs. concentration curves as shown in FIG. 5 was constructed.
No effect of polypeptide concentration versus concentration was seen. Concentration vs. time curves for each polypeptide are shown in FIG. Figure 6 shows that since the development of regions containing different concentrations of polypeptide proceeds along the same time course, the polypeptide-to-polypeptide ratio changes as development proceeds (assuming background concentration is subtracted). ) indicates the fact that it does not change drastically. As in the case of photographic development, the slope of the straight line portion of this development curve increases as the development time increases. Once maximum staining is achieved (8
(~12 minutes) Further development only increases the background level and eventually turns the entire gel black. In photography, it is possible to create a "gamma curve" which shows that the slope of the density/exposure relationship increases as the development time increases. FIG. 7 shows that the concentration/polypeptide concentration slope similarly increases with increasing development time for the silver-stained polypeptides of the present invention. The slope of the concentration/polypeptide relationship increases greatly for different polypeptides. The flat areas seen in this experiment are due to silver depletion and not developer depletion. This is because the application of developer does not cause further image formation. These findings for each purified polypeptide were reproduced in all gel patterns. When E. coli lysate patterns were photographed continuously during development and the concentrations of 25 types of polypeptide spots were monitored, as shown in Figure 8, the correlation coefficient of concentration vs. concentration was twice as high as the average polypeptide concentration. was greater than 0.90 during different times. There are difficulties when using silver staining quantitatively or quantitatively. Two of them are shown in FIG. A region of low concentration happens to be found in the center of the dense polypeptide spot. Rarely, a background region of lower density may be seen adjacent to a dark spot. Similar artifacts have been observed by early photographers who had to use thick emulsions that limited development and the efficiency of diffusion of fresh developer solution. By increasing the development time, such negative staining may become less noticeable and disappear, but the background becomes larger, the slope of the polypeptide concentration versus concentration relationship increases, and the linearity of the staining becomes less noticeable. There will be less space available. One type is negative staining. Staining saturation limits the quantifiable polypeptide concentration in a spot or band (generally higher than 2 ng/ ^mm2 ). A third problem occurs when a gel thicker than the thickness of the gel used (0.8 mm) is used. Separate images appear on each side of the slab gel. This may be due to the increased diffusion problem described above or due to the fact that there is a substantial difference in the distribution of polypeptides within such thick gels. Two-dimensional electrophoresis has become a valuable method in the fields of physiology and genetics because of its ability to separate hundreds of polypeptides in a single configuration. Quantitation of these polypeptides relies on accurate and sensitive microconcentration measurements of polypeptide spots whose concentration is precisely related to the concentration of the polypeptide. Methods for visualizing polypeptides having this regularity include autoradiography, several methods using organic dyes, and the silver staining method of the present invention described below. Successful quantitative analysis of these polypeptides depends on the selection of appropriate analytical methods for intersample comparisons. A number of polypeptides that are present in approximately constant amounts in a particular cell or specimen and are constitutively synthesized were utilized for the present invention. Such a polypeptide can be said to act as an internal standard. These constituent polypeptides are first used as internal markers of charge and molecular weight, and these parameters can be accurately evaluated for the polypeptide of interest and to create accurate pairings of polypeptide spots in different gels. can. After measurements are then taken with a densitometer, the constituent polypeptide concentrations are used to normalize the concentration obtained from a particular gel to the concentration found in a reference gel. Correlation coefficients between each of the 700 polypeptides and gels of similar samples are generally greater than 0.92. The average error between equivalent spots on the separation gel is 15-30%. Improvements in image visualization and quantitative densitometry are giving a third dimension to electrophoretic analytical techniques. FIG. 4 shows the same subsection of a lymphocyte lysate gel to illustrate the effect of using different amounts of polypeptide. The following amount of polypeptide was loaded onto each gel: 200 μg
(20μl), 100μg (10μl), 50μg (5μl), 25μg (
25 μl)
and 10 μg (1 μl). The total concentration (average concentration - background concentration) x area of each of the 45 polypeptide spots in each gel was compared with the corresponding spot concentration in each of the other gels. The correlation coefficient and slope were as follows.

【table】

【table】

【table】

[Table] Figure 5 shows concentration versus density curves for eight types of polypeptides. Concentrations are given in ng of polypeptide added per well. For comparison, polypeptide concentrations (same dilutions) are given in μg/mm ² in FIG. 4.
The background was subtracted after determining the mode density around each band using a histogram. FIG. 6 shows concentration versus development time curves for each polypeptide. Polypeptide concentrations are given here in μg/mm ² . FIG. 7 shows the change in the slope of the relationship between polypeptide and concentration when the development time was increased in sodium carbonate-formalin. The slope is 100 to 100% for 8 different purified polypeptides
Measured at 200ng. FIG. 8 shows the continuous development of E. coli lysate patterns. Lysate polypeptides were electrophoresed and images analyzed as described above. Concentration measurements were performed on 25 polypeptide spots.
The statistical data is as follows.

【table】

【table】

【table】

[Table] Figure 9 shows the artifacts seen with the silver staining of the present invention. This is a subregion of the lymphocyte lysate pattern. Concentration plots were made across the gel as indicated by the dotted line. The double peak is due to the polypeptide spot due to the low concentration inner region. Example 4 (Polypeptide Assay) Polypeptides were precipitated with potassium dichromate and then filtered under reduced pressure using cellulose nitrate paper. The source of this polypeptide was purified bovine serum albumin, and the amounts tested ranged from 0.1 μg to 100 μg. After exposure, the polypeptides were successfully stained according to the preferred method of the invention. Since no gel was used, the fixing step was omitted. However, it should be noted that the potassium dichromate used as an oxidizing agent in the photographic reversal process also acts as a fixing agent. The dye thus obtained is sufficiently sensitive that this embodiment of the staining method of the invention can be used as a quantitative polypeptide assay by comparing its staining intensity with that of a standard. It was showing. Example 5 (Tissue Staining) Thin tissue sections were successfully stained using the silver staining method of the present invention. This embodiment demonstrates that improved histological staining can be achieved using the methods of the invention. This embodiment is useful for biopsies. Example 6 (Optimal Dyeing Procedure) A dye was prepared according to the preferred embodiment of the invention as follows. Amounts are stated herein unless otherwise specified. (1) Fix the gel in trichloroacetic acid or methanol/acetic acid solution. (2) Wash three times with 10% ethanol-5% acetic acid solution. (3) Soak the gel in 0.0034M potassium dichromate and 0.0032N nitric acid for 5 minutes. (4) Soak the gel in 0.012M silver nitrate for 20 minutes. (5) Rinse the gel in a solution containing 0.28 M sodium carbonate and 0.5 ml formaldehyde with stirring. This last step requires changing the solution at least twice to avoid contaminating the gel surface with precipitated silver salts. (6) Stop the staining by washing the gel in 3% acetic acid for 5 minutes before background development. Except that active irradiation was not performed,
Up to this point is the staining operation as in the previous example. In an optimal embodiment of the invention, (7) Wash the gel two or more times for 5 minutes each with a 3% acetic acid solution. Furthermore, (8) repeat steps 4 to 6 at least once. Experiments using two-dimensional electrophoretic protein patterns from E. coli gels using the procedure described above were twice as sensitive for the first cycle (20 proteins were measured on each gel for each cycle). .

[Table] From the above, it can be seen that if steps 4 to 6 are repeated, the average protein spot concentration increases, which is preferable, but the average background also increases, which is not preferable. Therefore, steps 4 to 6 are preferably carried out 1 to 3 times,
Most preferably it should be done twice. As the average protein spot concentration increased, many proteins that were initially very faint became easily identified.

[Brief explanation of drawings]

Figure 1 shows the sensitivity of the staining of the present invention and the sweet tour.
Graph comparing Merrill and Shiffrin's (1979) histologically derived silver staining, 2nd
The figure shows a density radiograph of a two-dimensional electrophoresis gel containing polypeptides stained by the method of the present invention;
FIG. 3 is a composite radiograph showing an enlarged subarea of the gel shown in FIG. 2; FIG. 4 is a radiograph showing a comparison of staining according to the invention for various polypeptide concentrations; FIG. The figure shows a curve showing the relationship between the photographic density and the polypeptide concentration, FIG. 6 shows an X-ray of the curve showing the relationship between the photographic density and time for each polypeptide, and FIG. Curves showing that the slope of photographic density versus polypeptide concentration similarly increases as the development time increases for peptides; FIG. 8 is a curve showing the correlation coefficient of photographic density versus photographic density over time; FIG. The figure is an X-ray photograph showing artifacts that occur when using the silver stain of the present invention quantitatively or qualitatively.

Claims (1)

[Scope of Claims] 1. The following steps having no operating radiation other than ambient illumination: photoreversing a polypeptide gel by treatment with an oxidizing agent; and treating the polypeptide gel with a photosensitive salt. A method for silver staining polypeptides in a gel, comprising the steps of: forming a latent image of the dye by treating the polypeptide gel with a reducing agent, and developing the dye image by treating the polypeptide gel with a reducing agent. 2. A method according to claim 1, characterized in that the steps of forming a latent dye image and developing the dye image are repeated at least once. 3 The gel is made of at least one member of the group consisting of polyacrylamide, agarose or cellulose acetate.
the polypeptide is fixed by a dye fixing step, and the oxidizing agent is selected from at least one of the group consisting of acid dichromate, potassium permanganate, molecular oxygen, potassium ferricyanide, iodine, or quinone. and the photosensitive salt is selected from at least one of the group consisting of silver, gold, platinum, palladium or iridium photosensitive salts, and the reducing agent is a metal compound of iron, tungsten, vanadium and molybdenum,
3. A method according to claim 2, characterized in that it is selected from at least one of the group consisting of hydroquinone, pyrogallol, p-aminophenol, p-phenylenediamine, paraformaldehyde or formaldehyde. 4 The gel is polyacrylamide gel, the oxidizing agent is selected from the group consisting of dichromate and molecular oxygen, the photosensitive salt is silver salt, and the reducing agent is hydroquinone, pyrogallol, p-phenylenediamine, paraformaldehyde. or formaldehyde. 5. The method according to claim 4, wherein the oxidizing agent is an aqueous potassium dichromate solution containing a small amount of nitric acid, the photosensitive salt is silver nitrate, and the reducing agent is paraformaldehyde, formaldehyde, or a mixture thereof. Method. 6. Claim 3, characterized in that the fixing is carried out with a reagent selected from at least one of the group consisting of glutaraldehyde, mercury oxide, lead oxide, osmium oxide, formaldehyde, paraformaldehyde, trichloroacetic acid, or acetic acid. Method described. 7. Process according to claim 6, characterized in that the fixing agent is selected from the group consisting of trichloroacetic acid, formaldehyde, paraformaldehyde or acetic acid. 8. Process according to claim 7, characterized in that the fixing agent is acetic acid (approximately 5-20%), used in an aqueous mixture with methanol and/or ethanol (approximately 10-50%). 9. Fixing is carried out by dipping the polypeptide gel in a dye fixative 1 to 3 times for about 5 to 15 minutes each, and treatment with an oxidizing agent is carried out by dipping the fixed polypeptide gel therein for about 3 to 10 minutes. treatment with a photosensitive salt is performed by dipping the rinsed polypeptide gel therein for about 10 to 30 minutes, and treatment with a reducing agent is performed at least once therein. Claims 3 and 4, which are carried out by soaking for about 10 to 20 minutes.
The method according to any one of paragraphs 5 and 7. 10. The method according to claim 2, wherein staining is performed after the polypeptide in the gel is electrophoresed. 11. The following steps have no operating radiation other than ambient lighting: mixing the polypeptide with an aqueous solution of a fixative to form a polypeptide precipitate; passing the precipitate through paper; and oxidizing agent. a step of photographically reversing the polypeptide precipitate by treating the polypeptide precipitate with a photosensitive salt, a step of forming a latent dye image by treating the polypeptide precipitate with a photosensitive salt, and a step of treating the polypeptide precipitate with reduction to form a dye image. The polypeptide silver staining method in a gel according to claim 1, which comprises the step of developing. 12. The following processes that do not involve any operating radiation other than ambient illumination: photoreversal by treating the fixed tissue specimen with an oxidizing agent; and forming a latent image by treating the tissue with a photosensitive salt. A method for silver staining polypeptides in a gel according to claim 1, comprising the steps of: and developing a dye image by treating this tissue with a reducing agent. 13. The method of claim 2, wherein each step is repeated once.