CN113683724B - Polyacrylamide gel for human urine protein electrophoresis - Google Patents
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- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
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- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
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- G01N27/416—Systems
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Abstract
The invention belongs to the technical field of in-vitro diagnosis, and particularly provides polyacrylamide gel for human urine protein electrophoresis, which is characterized in that a certain amount of silane coupling agent is added into the polyacrylamide gel, and the selectable silane coupling agents comprise gamma-aminopropyl triethoxysilane, 3- (2, 3-epoxypropoxy) propyl trimethoxysilane and gamma-methacryloxypropyl trimethoxysilane so as to achieve the purposes of prolonging the gel preservation time and reducing the diffusion of a high-concentration sample. Compared with the existing polyacrylamide gel, the polyacrylamide gel provided by the invention is more suitable for typing and identifying human urine protein, has longer storage time, and is beneficial to large-scale clinical popularization and use.
Description
Technical Field
The invention belongs to the field of in-vitro diagnosis, and particularly relates to polyacrylamide gel for human urine protein electrophoresis.
Background
Urinary protein abnormalities are the most common manifestation and cause of renal disease. Currently, urine microalbumin (mAll), α 2-macroglobulin (α 2-MG), β 2-microglobulin (β 2-MG), uroretin-binding protein (URBP), N-acetyl- β -D-glucosaminidase (NAG), etc. are commonly used as urine protein detection methods in clinical practice. The type and the content of urine protein are closely related to the damage position and the damage degree of the kidney, and the comprehensive and comprehensive detection of the urine protein of a patient has important significance for the diagnosis and treatment of the kidney disease. The detection aims at a certain specific protein in urine, and has certain limitations in detection, such as susceptibility to influence of extra-renal factors, poor sensitivity, high price, and easy missed diagnosis and misdiagnosis particularly for early renal injury. In addition, early nephropathy (renal injury) caused by chronic diseases such as diabetes and hypertension has certain hiding property and reversibility, so people at high risk of nephropathy also need a noninvasive, high-sensitivity and high-specificity detection index for early detection and positioning of renal injury.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can separate proteins from each other according to the order of molecular weight, and is one of the most commonly used means for protein analysis and identification in biological laboratories. The urine of a nephropathy patient can be comprehensively analyzed by SDS-PAGE, physiological and pathological (glomerular, tubular and mixed) urine protein types can be distinguished, and the urine has higher sensitivity and specificity. In clinic, the kidney injury part can be accurately positioned, thereby being helpful for diagnosis and prognosis judgment of doctors.
Although SDS-PAGE gel products which can be stored for a long time and are used for scientific research are available in the market at present, human urine may also contain a large amount of impurities such as creatinine, urea, uric acid, inorganic salts and the like compared with conventional laboratory protein samples; urine samples of some nephropathy patients are often extremely high in protein concentration, and the condition that other samples are polluted by band diffusion occurs during electrophoresis, so that the operation difficulty of electrophoresis is increased, and the detection accuracy is interfered. For example, when a conventional SDS-PAGE preformed gel product faces interference of a large amount of complex creatinine, urea, uric acid and inorganic salts, the defects of band tailing, displacement and the like are generated, the accuracy of molecular weight analysis is interfered, and misjudgment of doctors is easily caused. Meanwhile, when the protein content in the sample is too high, the electrophoresis band is easy to diffuse transversely on the gel, and the adjacent lanes are interfered, so that a plurality of sample experiments fail.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides polyacrylamide gel suitable for human urine protein electrophoresis so as to achieve the purpose of improving the accuracy of a detection result. The invention specifically comprises the following contents:
the sodium dodecyl sulfate-polyacrylamide gel suitable for human urine electrophoresis is characterized in that the gel contains one of three silane coupling agents of gamma-aminopropyltriethoxysilane, 3- (2, 3-epoxypropoxy) propyltrimethoxysilane and gamma-methacryloxypropyltrimethoxysilane, and the content of the silane coupling agent in the gel is 3 wt%.
The polyacrylamide gel consists of concentrated gel and separation gel, and the formula of the concentrated gel is as follows:
30 380 ul of (w/v) premixed glue solution, 20 ul of 10 wt% ammonium persulfate, 4 ul of tetramethylethylenediamine, 90 ul of silane coupling agent, 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 1646 ul of deionized water, wherein the pH value is 7.2;
the formula of the separation gel is as follows:
30 2000 ul of premixed glue solution in percent (w/v), 75 ul of 10 wt percent ammonium persulfate, 5 ul of tetramethylethylenediamine, 240ul of silane coupling agent, 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 3380 ul of deionized water, wherein the pH value is 7.2;
the pre-mixed glue solution is prepared from acrylamide and methylene diacrylamide with the mass ratio of 29;
the silane coupling agent is one of gamma-aminopropyltriethoxysilane, 3- (2, 3-epoxypropoxy) propyltrimethoxysilane and gamma-methacryloxypropyltrimethoxysilane.
The preparation of the polyacrylamide gel comprises the following steps:
(1) Adding the prepared separating gel solution into a gel plate, adding ultrapure water to cover the surface of the gel plate, standing at room temperature for 20 min, and removing the ultrapure water on the upper layer of the separating gel;
(2) Adding the prepared concentrated gel into a gel plate, inserting a comb, standing for 30min, and forming gel after the gel is solidified;
(3) The prepared gel was stored in deionized water at 4 ℃.
Drawings
FIG. 1 shows the results of electrophoresis of a gel containing gamma-aminopropyltriethoxysilane as usual in example I.
FIG. 2 shows the electrophoresis results of the gel containing 3- (2, 3-glycidoxy) propyltrimethoxysilane.
FIG. 3 shows the result of electrophoresis of a gel of gamma-methacryloxypropyltrimethoxysilane obtained in example III.
FIG. 4 shows the result of electrophoresis of a conventional polyacrylamide gel in example four.
Detailed Description
It should be noted that, the contents described in the following embodiments do not limit the scope of the present invention, and all equivalent flow transformations made by the present specification or directly or indirectly applied to other related technical fields are included in the scope of the present invention. Unless otherwise specified, the chemicals used in the following examples were purchased from Sahn's chemical technology (Shanghai) Co., ltd.
The first embodiment is as follows: preparation of gel containing gamma-aminopropyltriethoxysilane
A polyacrylamide gel containing gamma-aminopropyltriethoxysilane was prepared as follows:
(1) Preparing a 30% (w/v) pre-mixed gel solution, weighing 2.9 g of acrylamide and 0.1 g of methylene diacrylamide, dissolving in 8ml of deionized water, slightly stirring until the acrylamide and the methylene diacrylamide are completely dissolved, and fixing the volume to 10 ml;
(2) Preparing 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution, weighing 13.08 g of bis (2-hydroxyethylamino) tris (hydroxymethyl) methane, dissolving in 40 ml of deionized water, adjusting the pH value to 7.2, and fixing the volume to 50 ml;
(3) Preparing 10 wt% ammonium persulfate solution, weighing 1g ammonium persulfate, dissolving in 9 ml deionized water, and storing at 4 ℃;
(4) Preparing separation glue, uniformly mixing 2000 ul of 30% (w/v) premixed glue solution, 3380 ul of deionized water, 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 240ul of gamma-aminopropyltriethoxysilane, quickly adding 75 ul of 10 wt% ammonium persulfate solution and 5 ul of tetramethylethylenediamine, slightly stirring, pouring the prepared separation glue into a glue plate, adding 2 ml of deionized water, and standing for 20 min;
(5) Preparing concentrated glue, namely uniformly mixing 380 ul of 30% (w/v) premixed glue solution, 1646 ul of deionized water, 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 90 ul of gamma-aminopropyltriethoxysilane; at the moment, the upper deionized water of the rubber plate is removed, 20 ul of 10 wt% ammonium persulfate solution and 4 ul of tetramethylethylenediamine are quickly added into the concentrated glue, then the concentrated glue is poured into the rubber plate, a comb is inserted, and the mixture is kept stand for 30 min;
(6) And (4) storing, namely storing the prepared polyacrylamide gel in deionized water at 4 ℃, wherein the prepared polyacrylamide gel can be stored for at least 6 months under the above conditions.
Example two: preparation of gel containing 3- (2, 3-epoxypropoxy) propyltrimethoxysilane
A polyacrylamide gel containing 3- (2, 3-glycidoxy) propyltrimethoxysilane was prepared as follows:
(1) Preparing a 30% (w/v) pre-mixed glue solution, weighing 2.9 g of acrylamide and 0.1 g of methylene diacrylamide, dissolving in 8ml of deionized water, slightly stirring until the acrylamide and the methylene diacrylamide are completely dissolved, and fixing the volume to 10 ml;
(2) Preparing 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution, weighing 13.08 g bis (2-hydroxyethylamino) tris (hydroxymethyl) methane, dissolving in 40 ml deionized water, adjusting the pH value to 7.2, and fixing the volume to 50 ml;
(3) Preparing 10 wt% ammonium persulfate solution, weighing 1g ammonium persulfate, dissolving in 9 ml deionized water, and storing at 4 ℃;
(4) Preparing a separation gel, uniformly mixing 2000 ul of 30% (w/v) premixed gel solution, 3380 ul of deionized water, 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 240ul of 3- (2, 3-epoxypropoxy) propyltrimethoxysilane, quickly adding 75 ul of 10 wt% ammonium persulfate solution and 5 ul of tetramethylethylenediamine, slightly stirring, pouring the prepared separation gel into a gel plate, adding 2 ml of deionized water, and standing for 20 min;
(5) Preparing concentrated glue, namely uniformly mixing 380 ul of 30% (w/v) premixed glue solution, 1646 ul of deionized water, 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 90 ul of 3- (2, 3-epoxypropoxy) propyltrimethoxysilane; at this time, the upper deionized water layer of the rubber plate is removed, 20 ul of 10 wt% ammonium persulfate solution and 4 ul of tetramethylethylenediamine are rapidly added into the concentrated glue, then the concentrated glue is poured into the rubber plate, a comb is inserted, and the mixture is kept stand for 30 min;
(6) And (4) storing, namely storing the prepared polyacrylamide gel in deionized water at 4 ℃, wherein the polyacrylamide gel can be stored for at least 6 months under the above conditions.
Example three: preparation of gel containing gamma-methacryloxypropyltrimethoxysilane
A polyacrylamide gel containing gamma-methacryloxypropyltrimethoxysilane was prepared as follows:
(1) Preparing a 30% (w/v) pre-mixed gel solution, weighing 2.9 g of acrylamide and 0.1 g of methylene diacrylamide, dissolving in 8ml of deionized water, slightly stirring until the acrylamide and the methylene diacrylamide are completely dissolved, and fixing the volume to 10 ml;
(2) Preparing 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution, weighing 13.08 g of bis (2-hydroxyethylamino) tris (hydroxymethyl) methane, dissolving in 40 ml of deionized water, adjusting the pH value to 7.2, and fixing the volume to 50 ml;
(3) Preparing 10 wt% ammonium persulfate solution, weighing 1g ammonium persulfate, dissolving in 9 ml deionized water, and storing at 4 ℃;
(4) Preparing separation glue, uniformly mixing 2000 ul of 30% (w/v) premixed glue solution, 3380 ul of deionized water, 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 240ul of gamma-methacryloxypropyltrimethoxysilane, rapidly adding 75 ul of 10 wt% ammonium persulfate solution and 5 ul of tetramethylethylenediamine, slightly stirring, pouring the prepared separation glue into a glue board, adding 2 ml of deionized water, and standing for 20 min;
(5) Preparing concentrated glue, namely uniformly mixing 380 ul of 30% (w/v) premixed glue solution, 1646 ul of deionized water, 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 90 ul of gamma-methacryloxypropyltrimethoxysilane; at this time, the upper deionized water layer of the rubber plate is removed, 20 ul of 10 wt% ammonium persulfate solution and 4 ul of tetramethylethylenediamine are rapidly added into the concentrated glue, then the concentrated glue is poured into the rubber plate, a comb is inserted, and the mixture is kept stand for 30 min;
(6) And (4) storing, namely storing the prepared polyacrylamide gel in deionized water at 4 ℃, wherein the polyacrylamide gel can be stored for at least 6 months under the above conditions.
Example four: conventional Polyacrylamide gel preparation
A conventional polyacrylamide gel was prepared as follows:
(1) Preparing a 30% (w/v) pre-mixed gel solution, weighing 2.9 g of acrylamide and 0.1 g of methylene diacrylamide, dissolving in 8ml of deionized water, slightly stirring until the acrylamide and the methylene diacrylamide are completely dissolved, and fixing the volume to 10 ml;
(2) Preparing 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution, weighing 13.08 g bis (2-hydroxyethylamino) tris (hydroxymethyl) methane, dissolving in 40 ml deionized water, adjusting the pH value to 7.2, and fixing the volume to 50 ml;
(3) Preparing 10 wt% ammonium persulfate solution, weighing 1g ammonium persulfate, dissolving in 9 ml deionized water, and storing at 4 ℃;
(4) Preparing separation glue, mixing 2000 ul of 30% (w/v) premixed glue solution, 3380 ul of deionized water and 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution uniformly, quickly adding 75 ul of 10 wt% ammonium persulfate solution and 5 ul of tetramethylethylenediamine, stirring slightly, pouring the prepared separation glue into a glue board, adding 2.24 ml of deionized water, and standing for 20 min;
(5) Preparing concentrated glue, mixing 380 ul of 30% (w/v) premixed glue solution, 1736 ul of deionized water and 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution uniformly; at the moment, the upper deionized water of the rubber plate is removed, 20 ul of 10 wt% ammonium persulfate solution and 4 ul of tetramethylethylenediamine are quickly added into the concentrated glue, then the concentrated glue is poured into the rubber plate, a comb is inserted, and the mixture is kept stand for 30 min;
(6) And (4) storing, namely storing the prepared polyacrylamide gel in deionized water at 4 ℃ for 15 days under the condition.
Example five: polyacrylamide gel electrophoresis for urine typing detection of nephropathy patients
The electrophoresis analysis of urine of renal patients was performed using the polyacrylamide gels prepared in examples one to four as follows:
(1) Urine samples were processed and GenScript was applied to each individual urine sample ® Adding 20 ul of 5X Sample Buffer into 80 ul of urine Sample, and incubating for 5min at 90 ℃;
(2) 1L GenScript was taken ® Adding Tris-MOPS-SDS Running Buffer into a liquid tank electrolyzed by an electrophoresis apparatus, taking out polyacrylamide gel from deionized water, carefully pulling out a comb, and fixing the comb in a plate tank of the electrophoresis apparatus;
(3) Adding 20 ul of processed urine sample into the glue hole left after the comb is pulled out, switching on a power supply, starting a 180V constant voltage mode, and starting electrophoresis;
(4) After electrophoresis, taking glue, dyeing and decoloring.
FIG. 1 shows the results of electrophoresis of a gel containing gamma-aminopropyltriethoxysilane as usual in example I; FIG. 2 shows the results of electrophoresis of a gel containing 3- (2, 3-glycidoxy) propyltrimethoxysilane; FIG. 3 shows the result of electrophoresis of a gel of gamma-methacryloxypropyltrimethoxysilane of example III; FIG. 4 shows the result of electrophoresis of a conventional polyacrylamide gel in example four.
It can be seen that when the urine protein of a patient is analyzed by conventional gel electrophoresis, the sample with high urine protein content can be diffused transversely and interfere with the adjacent lane (figure 4); the resolution of the gel containing the siloxane is obviously improved, and the lateral diffusion of the sample with high urine protein can be effectively prevented from influencing the results of adjacent lanes (figure 1, figure 2 and figure 3); the gelling effect using gamma-methacryloxypropyltrimethoxysilane was significantly better than that of 3- (2, 3-glycidoxy) propyltrimethoxysilane and gamma-aminopropyltriethoxysilane (FIG. 3).
Claims (1)
1. The polyacrylamide gel suitable for human urine protein electrophoresis is characterized by consisting of concentrated gel and separation gel, wherein the concentrated gel and the separation gel contain a certain amount of silane coupling agent; the concentrated glue comprises: 30 380 ul of (w/v) premixed glue solution, 20 ul of 10 wt% ammonium persulfate, 4 ul of tetramethylethylenediamine, 90 ul of silane coupling agent, 860 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 1646 ul of deionized water, wherein the pH value is 7.2; the separating gel comprises: 30 2000 ul of premixed glue solution in percent (w/v), 75 ul of 10 wt percent ammonium persulfate, 5 ul of tetramethylethylenediamine, 240ul of silane coupling agent, 2300 ul of 1.25M bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution and 3380 ul of deionized water, wherein the pH value is 7.2; the silane coupling agent is one of gamma-aminopropyltriethoxysilane, 3- (2, 3-epoxypropoxy) propyltrimethoxysilane and gamma-methacryloxypropyltrimethoxysilane; the premixed glue solution is prepared from acrylamide and methylene diacrylamide in a mass ratio of 29.
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Citations (5)
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| EP0324539A2 (en) * | 1988-01-12 | 1989-07-19 | Northeastern University | Improved capillary gel electrophoresis columns |
| CN102643379A (en) * | 2011-06-17 | 2012-08-22 | 京东方科技集团股份有限公司 | Quadripolymer, electronic ink microcapsules and preparation method thereof |
| CN103163194A (en) * | 2013-03-22 | 2013-06-19 | 天津大学 | Device and method for transferring and analyzing proteins in gel on line |
| CN106754873A (en) * | 2016-12-05 | 2017-05-31 | 四川大学 | DNA separating kits |
| CN108181369A (en) * | 2017-12-22 | 2018-06-19 | 重庆康萃医药科技有限公司 | A kind of polyacrylamide gel and its kit |
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2021
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0324539A2 (en) * | 1988-01-12 | 1989-07-19 | Northeastern University | Improved capillary gel electrophoresis columns |
| CN102643379A (en) * | 2011-06-17 | 2012-08-22 | 京东方科技集团股份有限公司 | Quadripolymer, electronic ink microcapsules and preparation method thereof |
| CN103163194A (en) * | 2013-03-22 | 2013-06-19 | 天津大学 | Device and method for transferring and analyzing proteins in gel on line |
| CN106754873A (en) * | 2016-12-05 | 2017-05-31 | 四川大学 | DNA separating kits |
| CN108181369A (en) * | 2017-12-22 | 2018-06-19 | 重庆康萃医药科技有限公司 | A kind of polyacrylamide gel and its kit |
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| Title |
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| "生物大分子毛细管电泳分离与预浓集方法研究2012.03.15;李晶晶;《中国优秀硕士学位论文全文数据库 基础科学辑》;20120315(第03期);第8页第1.4.2.3（2）部分，第17页第2.1部分，第19页第2.3.1部分，第21页第2.4.1部分，第28-29页第2.5部分 * |
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