CN116773637A - Method for detecting low-density lipoprotein in serum by improved gel electrophoresis - Google Patents
Method for detecting low-density lipoprotein in serum by improved gel electrophoresis Download PDFInfo
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- 238000002156 mixing Methods 0.000 claims description 19
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a method for detecting low-density lipoprotein in serum by improved gel electrophoresis, belonging to the technical field of biological detection. The method comprises the following steps: (1) serum staining; (2) gel tube preparation; (3) electrophoresis; and (4) quantitatively detecting. The gel electrophoresis system has good separation effect, high detection accuracy and high detection efficiency, and can accurately separate and quantitatively analyze the total amount of the low-density lipoprotein and each subtype thereof by adjusting the proportion of the separation gel to the concentrated gel, the content of the polyacrylamide in the gel liquid and the configuration of the staining liquid, so that the detection process is simple and easy to implement, the detection time is short, and the method has important significance for better judging the risk of cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a method for detecting low-density lipoprotein in serum by improved gel electrophoresis.
Background
Cardiovascular disease (CVD) is a general term for circulatory diseases, mainly including diseases of the heart, arteries, veins, capillaries, etc., and is generally closely related to atherosclerosis. Currently, CVD morbidity and mortality are still in the ascending phase and the leading mortality rate for various diseases. Thus, intervention in the high risk group of CVD can reduce the incidence of CVD. Dyslipidemia is one of important risk factors of CVD, so that developing blood lipid detection and standardization of measurement results plays an important role in preventing and treating CVD.
Lipids in human blood are substantially bound to proteins into lipoproteins, which can be classified into Very Low Density Lipoproteins (VLDL), low Density Lipoproteins (LDL), medium density lipoproteins (IDL), high Density Lipoproteins (HDL) and Chylomicrons (CM) according to their density. Low Density Lipoproteins (LDL) have heterogeneity and consist of a series of particles of varying size, density and chemical composition, which can be divided into up to 7 subtypes, named LDL-1, LDL-2, LDL-3, LDL-4, LDL-5, LDL-6 and LDL-7, respectively, depending on the diameter and density of the LDL particles. Wherein, large and light LDL (lbLDL) consists of LDL-1 and LDL-2 with particle diameter >27nm and density <1.03g/mL, which is defined as type A; small, dense LDL (sdLDL) consists of LDL-3 to LDL-7 with particle diameter <26nm, density >1.04g/mL, defined as form B. Experiments show that the level of sdLDL can be used as an index for predicting and evaluating the risk of cardiovascular diseases, and the level of LDL and each subfraction thereof can comprehensively and accurately reflect the real level of lipoprotein in patients with cardiovascular diseases, thereby providing possibility for early intervention and reducing the prevalence rate of cardiovascular diseases. Thus, lipoprotein profiling is of great importance in clinical diagnosis of disease. However, the heterogeneity of the low density lipoprotein subtypes makes it difficult to isolate and detect.
At present, the plasma lipoprotein detection has various methods, wherein the electrophoresis method has the advantages of high efficiency and practicability, is widely applied to lipoprotein separation and detection analysis, in particular to polyacrylamide gel electrophoresis, and the contained polyacrylamide gel has the dual effects of molecular sieve and charge separation, has higher resolution, can separate serum lipoprotein into a plurality of zones, and has the advantages of strong transparency, quick separation time, simple and convenient operation and the like. However, conventional gel systems have low resolution of plasma lipoproteins and poor separation. The reason is that the polyacrylamide gel is easy to hydrolyze, the preservation time is short, the gel system is easy to lose the separation effect, the hydrolysis shortens the migration distance of protein, and the resolution of protein bands is reduced. In order to ensure experimental effect, most of the prepared gel is used, and although the detection effect is good, the working efficiency is low and the time and labor cost is high.
Therefore, a gel electrophoresis system with good separation effect, high detection accuracy and high detection efficiency needs to be developed, so that each subtype in the low-density lipoprotein can be accurately separated and detected, and the real level of the lipoprotein can be comprehensively and accurately reflected.
Disclosure of Invention
Aiming at the problems, the invention provides an improved gel electrophoresis system with good separation effect, high detection accuracy and high detection efficiency, which can accurately separate and detect each subtype of the low-density lipoprotein in serum.
The invention is realized by the following technical scheme:
a method for detecting low density lipoprotein in serum by modified gel electrophoresis, comprising the following steps:
(1) Serum staining: taking a serum sample, adding a staining solution, uniformly mixing, then carrying out constant-temperature water bath, centrifuging, and taking a supernatant to obtain stained serum;
(2) Gel tube preparation: fixing a glass tube on a bracket, sequentially adding a second layer of separating glue, a first layer of separating glue and concentrated glue into the glass tube, adding a next glue solution after the glue solution is polymerized and leveled on the surface of the glue solution each time, and finally immersing the whole into a preservation solution to obtain a gel tube; the second layer of separating glue, the first layer of separating glue and the concentrated glue all comprise: polypropyleneAmides, H 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate;
(3) Electrophoresis: after removing the surface of the gel tube and the preservation solution in the tube, vertically fixing the gel tube in an electrophoresis tank, wherein a first layer of separation gel and concentrated gel are positioned at the negative end of the electrophoresis tank, a second layer of separation gel is positioned at the positive end of the electrophoresis tank, then respectively adding electrophoresis buffer solution into the electrophoresis tank, slowly adding dyed serum into the gel tube, covering the surface of the concentrated gel, and switching on a stabilized voltage power supply for electrophoresis after the solution in the system is stabilized;
(4) Quantitative detection: and after electrophoresis, taking down the gel tube, scanning the absorbance change of different positions in the first layer of separation gel and the second layer of separation gel of the gel tube by using a scanner, and quantitatively detecting the percentage of the low-density lipoprotein of different subtypes to the whole serum lipoprotein according to the curve obtained by the absorbance change of the low-density lipoprotein of different subtypes along with the position change.
As the optimization of the technical scheme, in the step (2), the volume ratio of the second layer of separation gel to the first layer of separation gel to the concentrated gel in the gel tube is 5-7:1-2:1.
Preferably, in the step (2), the second layer of the separating gel comprises polyacrylamide and H 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate in a volume ratio of 28-35:150-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration T of the second layer of separating gel is 3-4% (the gel concentration T is the mass sum of acrylamide and methylene bisacrylamide divided by the total volume), and the pH value is 8.5-9.
Preferably, in the step (2), the polyacrylamide and H in the first layer of separation gel 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate in a volume ratio of 45-50:200-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration T=4.5-6% and the pH value of the first layer of separating gel is 8.5-9.
Preferably, in the step (2), polyacrylamide and H in the concentrated gel 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfateThe volume ratio is 20-25:150-250:4.5-6:55-65:1-2:0.1-0.2:1; the gel concentration T=2-3% and the pH value of the concentrated gel is 6.5-7.
As the preferable choice of the technical scheme, in the step (2), the preservation solution is prepared by mixing sucrose, tris-HCl, glycerol and Proclin 300 according to a mass ratio of 9-11 g, 38-45 mL, 1mL, and 0.1-0.2 mL.
As the optimization of the technical scheme, in the step (2), distilled water is added on the liquid level immediately after each glue solution is added, the solution is kept stand for 30-40 min, the water layer is removed after the glue solution surface is polymerized and leveled, and then the next glue solution is added.
Preferably, in the step (1), the volume ratio of the serum sample to the staining solution is 8-10:1.
As the optimization of the technical scheme, in the step (1), the dyeing liquid is prepared from Sudan black B, absolute ethyl alcohol, dimethyl sulfoxide and ethylene glycol, wherein the concentration of the Sudan black B in the dyeing liquid is 2-3 g/L, and the volume ratio of the absolute ethyl alcohol to the dimethyl sulfoxide to the ethylene glycol is 1-2:1:1-2.
In the step (3), the buffer solution is prepared by mixing tris (hydroxymethyl) aminomethane and boric acid according to a mass ratio of 2-3:1.
Compared with the prior art, the invention has the advantages that:
1. the gel electrophoresis system has good separation effect, high detection accuracy and high detection efficiency, and can accurately separate and quantitatively analyze the total amount of the low-density lipoprotein and each subtype thereof by adjusting the proportion of the separation gel to the concentrated gel, the content of the polyacrylamide in the gel liquid and the configuration of the staining liquid, so that the real level of the lipoprotein can be comprehensively and accurately reflected, the detection process is simple and easy to implement, the detection time is short, the detection process only needs about 75 minutes, and the method has important significance for better judging the cardiovascular and cerebrovascular disease risk.
2. The gel separation column is divided into a second layer of separation gel, a first layer of separation gel and a concentrated gel, wherein the contents of polyacrylamide contained in each gel solution are different, and three kinds of polyacrylamide with different contents are used as electrophoresis carriers to match with lipoproteins with different particles and densities, so that quantitative and qualitative analysis of each subtype of lipoproteins is more accurate. Wherein, the main function of the second layer of separating gel and the first layer of separating gel is to separate each subtype of lipoprotein, the function of the concentrated gel is to play a role of accumulation, and all the subtypes of lipoprotein are compressed in a narrow zone, thereby improving the sensitivity of electrophoresis.
3. The concentration of the second layer of separation gel is set according to the pore diameter of the gel, and the diameter of the separated lipoprotein particles is larger (more than 15 nm), so that the gel concentration T is required to be set below 4%, and the low gel concentration can enable the pore diameter of the gel to be large, so that the lipoprotein particles can be better separated, and the detection effect is improved.
4. The dyeing liquid is prepared from sudan black B, absolute ethyl alcohol, dimethyl sulfoxide and ethylene glycol, and has good solubility, dispersibility and stability and long shelf life of up to 1 year. Dimethyl sulfoxide is used as a solvent, and mainly plays a role in dissolution; the absolute ethyl alcohol is used as a dispersing agent, so that lipoprotein in serum has good dispersibility, and the resolution ratio of electrophoresis detection is improved; the ethylene glycol is used as a densifier, so that the density of the staining solution can be increased, a stained serum sample can be gathered and sunk during electrophoresis detection, and the sample is prevented from being dispersed into the buffer solution during electrophoresis. Through the combined action of all components in the staining solution, the combination of Sudan black B and all subtypes of lipoprotein can be promoted to be uniform, the staining result is uniform, the effect and the efficiency of electrophoresis detection are improved, and therefore all subtypes of lipoprotein can be qualitatively detected well at the same time.
Drawings
FIG. 1 is a gel electrophoresis pattern obtained by electrophoresis of serum from a patient diagnosed with cardiovascular disease according to the method of example 1.
FIG. 2 is a gel electrophoresis pattern obtained by electrophoresis of serum of a healthy person according to the method of example 1.
FIG. 3 is a gel electrophoresis pattern obtained by electrophoresis of serum from a patient diagnosed with cardiovascular disease according to the method of comparative example 1.
FIG. 4 is a gel electrophoresis pattern obtained by electrophoresis of serum of a healthy person according to the method of comparative example 1.
Detailed Description
The present invention is further illustrated by the following examples, which are only intended to illustrate the present invention and not to limit the scope of the present invention.
Example 1
A method for detecting low density lipoprotein in serum by modified gel electrophoresis, comprising the following steps:
(1) Serum staining: collecting a venous blood sample after 12h of empty stomach, separating serum out in 2h, taking 120 mu L of serum, adding 15 mu L of staining solution, uniformly mixing, then carrying out constant-temperature water bath at 37 ℃ for 30min, centrifuging at a rotating speed of 3000r/min for 10min, and taking supernatant after centrifuging to obtain stained serum;
the preparation of the staining solution comprises the following steps: preparing Sudan black B into 3g/L, adding 10mL of absolute ethyl alcohol, 10mL of dimethyl sulfoxide and 10mL of ethylene glycol, uniformly mixing, heating at 40 ℃ for 10min, continuously stirring during heating, filtering with a filter membrane with the pore diameter of 0.22 mu m while the mixture is hot, and filtering once again after cooling to obtain a dyeing liquid;
(2) Gel tube preparation: fixing a glass tube on a bracket, sequentially adding 1050 mu L of second layer of separating gel, 270 mu L of first layer of separating gel and 160 mu L of concentrated gel into the glass tube, immediately adding 50 mu L of distilled water on the liquid surface after each gel solution is added, standing for 30min to isolate air and make the surface of the gel solution smooth, removing a water layer after polymerization of the surface of the gel solution is smooth, then adding the next gel solution, and finally immersing the whole into 500mL of preservation solution to obtain a gel tube;
the preparation of the second layer of separating glue comprises the following steps: 1.480mL of 30% polyacrylamide (Acr: bis=29:1) and H were taken 2 O12.3592 mL, 1mol/L Tris-HCl (pH 8.9) 380 mu L, 25% sucrose 480 mu L, 25% glycerol 48 mu L, tetramethyl ethylenediamine 4.8 mu L and 10% ammonium persulfate 48 mu L are uniformly mixed to prepare a second layer of separating gel with gel concentration T=3.0 and pH 8.6 and 14.8mL system;
the preparation of the first layer of separating glue comprises the following steps: 3.420mL of 30% polyacrylamide (Acr: bis=29:1) and H were taken 2 O 16.558mL, 550. Mu.L of 1mol/L Tris-HCl (pH 8.9), 700. Mu.L of 25% sucrose, 70. Mu.L of 25% glycerol, 7.0. Mu.L of tetramethyl ethylenediamine and 70. Mu.L of 10% ammonium persulfate are uniformly mixed to prepare a first layer of separation gel with the gel concentration of T=4.8% and the pH of 8.6 and 21.375 mL;
the preparation of the concentrated glue comprises the following steps: 30% polyacrylamide (Acr: bis=29:1) was taken as 445.0 μ L, H 2 O4127.2 mu L, 1mol/L Tris-HCl (pH 6.8) 90 mu L, 25% sucrose 1100 mu L, 25% glycerol 18 mu L, tetramethyl ethylenediamine 1.8 mu L and 10% ammonium persulfate 18 mu L are uniformly mixed to prepare concentrated gel with gel concentration T=2.3% and pH 6.9 and 5.8 mL;
the preparation of the preservation solution comprises the following steps: dissolving 90mL of 1.5mol/L Tris-HCl (pH 6.7), 22g of sucrose, 2.0mL of glycerol and 300 280 mu L of Proclin in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a preservation solution;
(3) Electrophoresis: after removing the surface of the gel tube and the preservation solution in the tube, vertically fixing the gel tube in an electrophoresis tank, wherein a first layer of separation gel and a concentrated gel are positioned at the negative end of the electrophoresis tank, a second layer of separation gel is positioned at the positive end of the electrophoresis tank, then adding 600mL of electrophoresis buffer solution into the electrophoresis tank respectively, slowly adding 40 mu L of dyed serum into the gel tube, covering the surface of the concentrated gel, and switching on a stabilized voltage power supply after the solution in the system is stable, wherein the current is 2 mA/tube, and the electrophoresis time is 80min;
the preparation of the electrophoresis buffer solution comprises the following steps: dissolving 12.025g of tris and 5.8g of boric acid in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain an electrophoresis buffer solution;
(4) Quantitative detection: and after electrophoresis, taking down the gel tube, scanning the absorbance change of different positions in the first layer of separation gel and the second layer of separation gel of the gel tube by using a scanner, and quantitatively detecting the percentage of the low-density lipoprotein of different subtypes to the whole serum lipoprotein according to the curve obtained by the absorbance change of the low-density lipoprotein of different subtypes along with the position change.
Example 2
A method for detecting low density lipoprotein in serum by modified gel electrophoresis, comprising the following steps:
(1) Serum staining: collecting a venous blood sample after 12h of empty stomach, separating serum out in 2h, taking 150 mu L of serum, adding 18 mu L of staining solution, uniformly mixing, then carrying out constant-temperature water bath at 37 ℃ for 30min, centrifuging at a rotating speed of 3000r/min for 10min, and taking supernatant after centrifuging to obtain stained serum;
the preparation of the staining solution comprises the following steps: preparing Sudan black B into 2g/L, adding 15mL of absolute ethyl alcohol, 12mL of dimethyl sulfoxide and 15mL of ethylene glycol, uniformly mixing, heating at 40 ℃ for 12min, continuously stirring during heating, filtering with a filter membrane with the pore diameter of 0.22 mu m while the mixture is hot, and filtering once again after cooling to obtain a dyeing liquid;
(2) Gel tube preparation: fixing a glass tube on a bracket, sequentially adding 1065 mu L of second layer of separating glue, 280 mu L of first layer of separating glue and 175 mu L of concentrated glue into the glass tube, immediately adding 50 mu L of distilled water on the liquid surface after each glue solution is added, standing for 30min to isolate air and make the glue solution surface smooth, removing a water layer after the glue solution surface is polymerized and smooth, then adding the next glue solution, and finally immersing the whole into 500mL of preservation solution to obtain a gel tube;
the preparation of the second layer of separating glue comprises the following steps: 1.455mL of 30% polyacrylamide (Acr: bis=29:1), H was taken 2 O11.624 mL, 1mol/L Tris-HCl (pH 8.9) 400 μL, 25% sucrose 500 μL, 25% glycerol 50 μL, tetramethyl ethylenediamine 5 μL and 10% ammonium persulfate 46 μL are uniformly mixed to prepare a second layer of separating gel with gel concentration T=3.1% and pH 8.8 and 14.08mL system;
the preparation of the first layer of separating glue comprises the following steps: 3.460mL of 30% polyacrylamide (Acr: bis=29:1) and H were taken 2 O17.613 mL, 1mol/L Tris-HCl (pH 8.9) 580 μL, 25% sucrose 750 μL, 25% glycerol 80 μL, tetramethyl ethylenediamine 7.0 μL and 10% ammonium persulfate 70 μL are uniformly mixed to prepare a first layer of separating gel with gel concentration T=4.6 and pH 8.7 and 22.56 mL;
the preparation of the concentrated glue comprises the following steps: 30% polyacrylamide (Acr: bis=29:1) 465.2 μ L, H was taken 2 O3871.8. Mu.L, 1mol/L Tris-HCl (pH 6.8) 95. Mu.L, 25% sugarcaneMu L of sugar 1120, 25 mu L of 25% glycerol, 3.0 mu L of tetramethyl ethylenediamine and 20 mu L of 10% ammonium persulfate are uniformly mixed to prepare concentrated gel with gel concentration T=2.5% and pH of 6.7 and 5.6 mL;
the preparation of the preservation solution comprises the following steps: dissolving 65mL of 1.5mol/L Tris-HCl (pH 6.7), 15g of sucrose, 1.5mL of glycerol and 300 250 mu L of Proclin in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a preservation solution;
(3) Electrophoresis: after removing the surface of the gel tube and the preservation solution in the tube, vertically fixing the gel tube in an electrophoresis tank, wherein a first layer of separation gel and a concentrated gel are positioned at the negative end of the electrophoresis tank, a second layer of separation gel is positioned at the positive end of the electrophoresis tank, then adding 600mL of electrophoresis buffer solution into the electrophoresis tank respectively, slowly adding 40 mu L of dyed serum into the gel tube, covering the surface of the concentrated gel, and switching on a stabilized voltage power supply after the solution in the system is stable, wherein the current is 2 mA/tube, and the electrophoresis time is 80min;
the preparation of the electrophoresis buffer solution comprises the following steps: dissolving 14.120g of tris and 6.0g of boric acid in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain an electrophoresis buffer solution;
(4) Quantitative detection: and after electrophoresis, taking down the gel tube, scanning the absorbance change of different positions in the first layer of separation gel and the second layer of separation gel of the gel tube by using a scanner, and quantitatively detecting the percentage of the low-density lipoprotein of different subtypes to the whole serum lipoprotein according to the curve obtained by the absorbance change of the low-density lipoprotein of different subtypes along with the position change.
Example 3
A method for detecting low density lipoprotein in serum by modified gel electrophoresis, comprising the following steps:
(1) Serum staining: collecting a venous blood sample after 12h of empty stomach, separating out serum in 2h, taking 180 mu L of serum, adding 20 mu L of staining solution, uniformly mixing, then carrying out constant-temperature water bath at 37 ℃ for 30min, centrifuging at a rotating speed of 3000r/min for 10min, and taking supernatant after centrifuging to obtain stained serum;
the preparation of the staining solution comprises the following steps: preparing Sudan black B into 3g/L, adding 15mL of absolute ethyl alcohol, 10mL of dimethyl sulfoxide and 15mL of ethylene glycol, uniformly mixing, heating at 40 ℃ for 10min, continuously stirring during heating, filtering with a filter membrane with the pore diameter of 0.22 mu m while the mixture is hot, and filtering once again after cooling to obtain a dyeing liquid;
(2) Gel tube preparation: fixing a glass tube on a bracket, sequentially adding 1250 mu L of second layer of separating glue, 300 mu L of first layer of separating glue and 220 mu L of concentrated glue into the glass tube, immediately adding 50 mu L of distilled water on the liquid surface after each glue solution is added, standing for 30min to isolate air and make the glue solution surface smooth, removing a water layer after the glue solution surface is polymerized and smooth, then adding the next glue solution, and finally immersing the whole into 500mL of preservation solution to obtain a gel tube;
the preparation of the second layer of separating glue comprises the following steps: 1.510mL of 30% polyacrylamide (Acr: bis=29:1) and H were taken 2 O9.749 mL, 1mol/L Tris-HCl (pH 8.9) 400 μL, 25% sucrose 470 μL, 25% glycerol 60 μL, tetramethyl ethylenediamine 6.0 μL and 10% ammonium persulfate 45 μL are uniformly mixed to prepare a second layer of separating gel with gel concentration T=3.7 and pH 8.9 and 12.24 mL;
the preparation of the first layer of separating glue comprises the following steps: 3.230mL of 30% polyacrylamide (Acr: bis=29:1) and H were taken 2 O15.646 mL, 1mol/L Tris-HCl (pH 8.9) 500 μL, 25% sucrose 650 μL, 25% glycerol 80 μL, tetramethyl ethylenediamine 9.0 μL and 10% ammonium persulfate 65 μL are uniformly mixed to prepare a first layer of separating gel with gel concentration T=4.8% and pH 8.5 and 20.18 mL;
the preparation of the concentrated glue comprises the following steps: 30% polyacrylamide (Acr: bis=29:1) was taken as 445.0 μ L, H 2 O3977 mu L, 1mol/L Tris-HCl (pH 6.8) 100 mu L, 25% sucrose 1230 mu L, 25% glycerol 25 mu L, 3.0 mu L of tetramethyl ethylenediamine and 20 mu L of 10% ammonium persulfate are uniformly mixed to prepare concentrated gel with gel concentration T=2.3% and pH 6.9 and 5.8 mL;
the preparation of the preservation solution comprises the following steps: dissolving 100mL of 1.5mol/L Tris-HCl (pH 6.7), 25g of sucrose, 2.3mL of glycerol and 300 320 mu L of Proclin in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a preservation solution;
(3) Electrophoresis: after removing the surface of the gel tube and the preservation solution in the tube, vertically fixing the gel tube in an electrophoresis tank, wherein a first layer of separation gel and a concentrated gel are positioned at the negative end of the electrophoresis tank, a second layer of separation gel is positioned at the positive end of the electrophoresis tank, then adding 600mL of electrophoresis buffer solution into the electrophoresis tank respectively, slowly adding 40 mu L of dyed serum into the gel tube, covering the surface of the concentrated gel, and switching on a stabilized voltage power supply after the solution in the system is stable, wherein the current is 2 mA/tube, and the electrophoresis time is 80min;
the preparation of the electrophoresis buffer solution comprises the following steps: dissolving 13.224g of tris and 5.5g of boric acid in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain an electrophoresis buffer solution;
(4) Quantitative detection: and after electrophoresis, taking down the gel tube, scanning the absorbance change of different positions in the first layer of separation gel and the second layer of separation gel of the gel tube by using a scanner, and quantitatively detecting the percentage of the low-density lipoprotein of different subtypes to the whole serum lipoprotein according to the curve obtained by the absorbance change of the low-density lipoprotein of different subtypes along with the position change.
Comparative example 1
Comparative example 1 differs from example 1 in that the separation gel, the concentrated gel and the running buffer in comparative example 1 are different from example 1, and the remaining detection methods are the same as in example 1. The preparation of the separation gel, the concentration gel and the running buffer in comparative example 1 is as follows:
the preparation of the separating gel comprises the following steps: 30% polyacrylamide (Acr: bis=29:1) 0.667mL, H was taken 2 O4.078 mL, 1mol/LTris-HCl (pH 8.9) 200 mu L, tetramethyl ethylenediamine 5.0 mu L and 10% ammonium persulfate 50 mu L are uniformly mixed to prepare a separating gel with gel concentration T=4.0%, pH 8.9 and pH 5.0 mL;
the preparation of the concentrated glue comprises the following steps: 30% polyacrylamide (Acr: bis=29:1) was taken at 300.0 μ L, H 2 O2.567 mL, 1mol/LTris-HCl (pH 6.8) 100 μl, tetramethyl ethylenediamine 3.0 μl and 10% ammonium persulfate 30 μl are mixed uniformly to prepare concentrated gel with gel concentration T=3.0%, pH 6.9 and pH 3.0 mL;
the electrophoretic buffer is prepared by the following steps: dissolving 3.3g of tris and 14.4g of glycine in water, uniformly mixing, fixing the volume to 1000mL, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain an electrophoresis buffer solution.
Evaluation of detection Effect
Serum from a clinically confirmed cardiovascular patient and serum from a healthy person were taken, each serum was subjected to electrophoresis in the same manner as in example 1 and comparative example 1, and after electrophoresis, the resultant electropherograms were scanned using an optical density scanner as shown in FIGS. 1 to 4. Wherein FIGS. 1 and 2 are gel electrophoresis patterns obtained by electrophoresis of serum of a patient diagnosed with cardiovascular disease and serum of a healthy person according to the method of example 1, respectively, and FIGS. 3 and 4 are gel electrophoresis patterns obtained by electrophoresis of serum of a patient diagnosed with cardiovascular disease and serum of a healthy person according to the method of comparative example 1, respectively.
As can be seen from FIGS. 1 and 2, the gel electrophoresis patterns of FIGS. 1 and 2 have high definition and resolution, and the method of example 1 is capable of clearly separating each subtype of low density lipoprotein, and has high detection accuracy. As can be seen from fig. 3 and 4, the gel electrophoresis patterns of fig. 3 and 4 have lower definition and resolution, which indicates that the separation effect of the method of comparative example 1 is poor.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method for detecting low density lipoprotein in serum by modified gel electrophoresis, which is characterized by comprising the following steps:
(1) Serum staining: taking a serum sample, adding a staining solution, uniformly mixing, then carrying out constant-temperature water bath, centrifuging, and taking a supernatant to obtain stained serum;
(2) Gel tube preparation: fixing the glass tube on the bracket, sequentially adding a second layer of separating glue, a first layer of separating glue and concentrated glue into the glass tube, adding the next glue solution after the glue solution surface is polymerized and leveled each time, and finally integratingImmersing in the preservation solution to obtain a gel tube; the second layer of separating glue, the first layer of separating glue and the concentrated glue all comprise: polyacrylamide, H 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate;
(3) Electrophoresis: after removing the surface of the gel tube and the preservation solution in the tube, vertically fixing the gel tube in an electrophoresis tank, wherein a first layer of separation gel and concentrated gel are positioned at the negative end of the electrophoresis tank, a second layer of separation gel is positioned at the positive end of the electrophoresis tank, then respectively adding electrophoresis buffer solution into the electrophoresis tank, slowly adding dyed serum into the gel tube, covering the surface of the concentrated gel, and switching on a stabilized voltage power supply for electrophoresis after the solution in the system is stabilized;
(4) Quantitative detection: and after electrophoresis, taking down the gel tube, scanning the absorbance change of different positions in the first layer of separation gel and the second layer of separation gel of the gel tube by using a scanner, and quantitatively detecting the percentage of the low-density lipoprotein of different subtypes to the whole serum lipoprotein according to the curve obtained by the absorbance change of the low-density lipoprotein of different subtypes along with the position change.
2. The method for detecting low-density lipoprotein in serum by modified gel electrophoresis according to claim 1, wherein in the step (2), the volume ratio of the second layer of separation gel, the first layer of separation gel and the concentrated gel in the gel column is 5 to 7:1 to 2:1.
3. The method for detecting low-density lipoprotein in serum by modified gel electrophoresis according to claim 2, wherein in step (2), polyacrylamide, H in the second layer of separation gel 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate in a volume ratio of 28-35:150-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration T=3-4% and pH value of the second layer separating gel is 8.5-9.
4. The method for detecting low density lipoprotein in serum by modified gel electrophoresis of claim 2 in which in step (2) the first layer of separation gel comprises polypropyleneEnamides, H 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate in a volume ratio of 45-50:200-300:7-9:9-11:1-2:0.1-0.2:1; the gel concentration T=4.5-6% and the pH value of the first layer of separating gel is 8.5-9.
5. The method for detecting low-density lipoprotein in serum by modified gel electrophoresis according to claim 2, wherein in step (2), polyacrylamide, H in the concentrated gel 2 O, tris-HCl, sucrose, glycerol, tetramethyl ethylenediamine and ammonium persulfate in a volume ratio of 20-25:150-250:4.5-6:55-65:1-2:0.1-0.2:1; the gel concentration T=2-3% and the pH value of the concentrated gel is 6.5-7.
6. The method for detecting low-density lipoprotein in serum by improved gel electrophoresis according to claim 1, wherein in the step (2), the preservation solution is prepared by mixing sucrose, tris-HCl, glycerol and Proclin 300 according to a plastid ratio of 9-11 g:38-45 ml:1 ml:0.1-0.2 mL.
7. The method for detecting low-density lipoprotein in serum by improved gel electrophoresis according to claim 1, wherein in the step (2), distilled water is added on the liquid surface immediately after each glue solution is added, the mixture is left stand for 30 to 40 minutes, the water layer is removed after the glue solution surface is polymerized and leveled, and then the next glue solution is added.
8. The method for detecting low-density lipoprotein in serum by modified gel electrophoresis according to claim 1, wherein in the step (1), the volume ratio of the serum sample to the staining solution is 8 to 10:1.
9. The method for detecting low-density lipoprotein in serum by improved gel electrophoresis according to claim 8, wherein in the step (1), the staining solution is prepared from sudan black B, absolute ethyl alcohol, dimethyl sulfoxide and ethylene glycol, the concentration of sudan black B in the staining solution is 2-3 g/L, and the volume ratio of absolute ethyl alcohol, dimethyl sulfoxide and ethylene glycol is 1-2:1:1-2.
10. The method for detecting low-density lipoprotein in serum by improved gel electrophoresis according to claim 1, wherein in the step (3), the buffer solution is prepared by mixing tris and boric acid according to a mass ratio of 2-3:1.
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