CN109270064B - Immunoprecipitation reagent based on dextran microspheres and preparation method and application thereof - Google Patents
Immunoprecipitation reagent based on dextran microspheres and preparation method and application thereof Download PDFInfo
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- CN109270064B CN109270064B CN201811336159.5A CN201811336159A CN109270064B CN 109270064 B CN109270064 B CN 109270064B CN 201811336159 A CN201811336159 A CN 201811336159A CN 109270064 B CN109270064 B CN 109270064B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Abstract
The invention provides an immunoprecipitation reagent based on dextran microspheres and a preparation method and application thereof, belonging to the technical field of biomedicine. The preparation method of the immunoprecipitation reagent based on the dextran microspheres, provided by the invention, uses the dextran particles with uniform particle size, is convenient to obtain materials, simple in preparation process of the microspheres, capable of realizing large-scale production and low in price; through a simple process, a large amount of immunoprecipitation reagents with good experimental effect can be rapidly prepared; the prepared immunoprecipitation reagent based on the dextran microspheres is applied in experiments, the result is accurate and reliable, and the method has good popularization and application values.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an immunoprecipitation reagent based on dextran microspheres and a preparation method and application thereof.
Background
Immunoprecipitation (Immunoprecipitation) is a classical method used to study protein-protein, protein-DNA, and protein-RNA interactions in the life science research process, and is also an effective method to determine the interaction of a target protein with a target molecule under physiological conditions. The principle is that the specific binding of antibody and target molecule is utilized, then protein A/G which specifically adsorbs Fc domain of IgG is added, and the protein A/G is coupled on the surface of solid phase carrier, and the separation of immune complex is carried out by centrifugation or magnetic separator. Therefore, the method provides technical support for describing a mutual regulation network in the life process.
Immunoprecipitation is also one of the methods currently indispensable for antibody purification. In the method, protein A/G is covalently coupled to a solid phase medium, such as Agarose beads or Magnetic beads, and when ascites or hybridoma cell supernatant is enriched from the solid phase medium, the antibody is combined with the protein A/G, so that the aim of enriching the antibody is fulfilled.
At present, protein A/G mainly takes Agarose beads or Magnetic beads as a solid-phase medium, and the preparation process is relatively complicated, the particle size is not uniform, the pressure change during separation is large, the separation effect is poor, and the price is high.
Disclosure of Invention
The invention aims to provide a preparation method of an immunoprecipitation reagent based on dextran microspheres, which can rapidly prepare a solid phase medium with uniform particle size through a simple preparation process, and has cheap raw materials.
The second purpose of the invention is to provide an immunoprecipitation reagent based on dextran microspheres, which has uniform particle size and avoids the inconsistency of particle size caused by large pressure change during separation.
The third purpose of the invention is to provide the application of the immunoprecipitation reagent based on the glucan microsphere in an immunoprecipitation test.
In order to achieve the above purpose of the invention, the following technical scheme is adopted:
a preparation method of an immunoprecipitation reagent based on dextran microspheres comprises the following steps:
washing glucan, adding water for dissolving, and performing ultrasonic dispersion to obtain glucan suspension;
mixing the glucan suspension with carbodiimide, adjusting the pH to 4.3-4.6, performing first incubation, centrifuging and washing to obtain glucan microsphere precipitate;
re-suspending the glucan microsphere precipitate, mixing the glucan microsphere precipitate with the recombinant protein, adjusting the pH value to 4.3-4.6, performing second incubation and centrifuging to obtain a glucan microsphere-recombinant protein precipitate;
washing the dextran microsphere-recombinant protein precipitate with PBS buffer solution, then resuspending to obtain a dextran microsphere-recombinant protein compound, and preparing the immunoprecipitation reagent based on the dextran microsphere.
The immunoprecipitation reagent based on the glucan microsphere is prepared by the preparation method of the immunoprecipitation reagent based on the glucan microsphere.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
Compared with the prior art, the invention has the beneficial effects that: the preparation method of the immunoprecipitation reagent based on the dextran microspheres, provided by the invention, uses the dextran particles with uniform particle size, is convenient to obtain materials, simple in preparation process of the microspheres, capable of realizing large-scale production and low in price; through a simple process, a large amount of immunoprecipitation reagents with good experimental effect can be rapidly prepared; the prepared immunoprecipitation reagent based on the dextran microspheres is applied in experiments, the result is accurate and reliable, and the method has good popularization and application values.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a comparison of dextran microspheres and protein A/G before and after coupling provided by an embodiment of the present invention;
FIG. 2 is a diagram showing results of a western blot experiment before and after sample purification according to an experimental example of the present invention;
FIG. 3 is a graph of the result of immunoprecipitation experiments on dextran microspheres-recombinant protein complexes provided in the experimental examples of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following describes an immunoprecipitation reagent based on dextran microspheres, a preparation method and an application thereof in an embodiment of the invention.
A preparation method of an immunoprecipitation reagent based on dextran microspheres comprises the following steps:
washing glucan, adding water for dissolving, and performing ultrasonic dispersion to obtain glucan suspension;
mixing the glucan suspension with carbodiimide, adjusting the pH to 4.3-4.6, performing first incubation, centrifuging and washing to obtain glucan microsphere precipitate;
re-suspending the glucan microsphere precipitate, mixing the glucan microsphere precipitate with the recombinant protein, adjusting the pH value to 4.3-4.6, performing second incubation and centrifuging to obtain a glucan microsphere-recombinant protein precipitate;
washing the dextran microsphere-recombinant protein precipitate with PBS buffer solution, then resuspending to obtain a dextran microsphere-recombinant protein compound, and preparing the immunoprecipitation reagent based on the dextran microsphere.
The dextran in the experiment can be purchased or made by oneself; yeast glucan was selected and obtained by homemade in this experiment.
Further, in a preferred embodiment of the present invention, the recombinant protein comprises one of protein A/G, protein A/G-fluorophore or secondary antibody.
Further, in a preferred embodiment of the invention, the fluorophore is one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, or NED.
Further, in the preferred embodiment of the present invention, the time for ultrasonic dispersion is 27-36 min.
Through ultrasonic dispersion, the glucan particles agglomerated into clusters can be dispersed to form a uniform glucan solution, which is beneficial to the subsequent experiments.
Further, in the preferred embodiment of the present invention, the pH is adjusted to 4.3-4.6 and HCl is selected for the first incubation; the first incubation was incubated for 15-20h at room temperature.
Further, in a preferred embodiment of the invention, the pH is adjusted to 4.3-4.6 and the second incubation is adjusted with NaOH and the second incubation is incubated at room temperature for 4-8 h.
Further, in the preferred embodiment of the present invention, the temperature of the second shaking culture is 20-35 ℃ and the culture time is 68-75 h.
Further, in the preferred embodiment of the present invention, the rotation speed of the dextran microsphere precipitate obtained by centrifugation and washing is 4700-5200rpm for 100-150 s.
Further, in a preferred embodiment of the present invention, after washing the dextran microsphere-recombinant protein precipitate, PBS buffer containing 0.02% sodium azide was selected for resuspension.
The prepared immunoprecipitation reagent based on dextran microspheres can be stably stored by using PBS buffer solution containing sodium azide for resuspension.
The immunoprecipitation reagent based on the glucan microsphere is prepared by the preparation method of the immunoprecipitation reagent based on the glucan microsphere.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a preparation method of an immunoprecipitation reagent based on dextran microspheres, which comprises the following steps:
preparation of yeast glucan:
1.1 adding 100g of high-activity yeast into 1L of NaOH (1M/L), and heating and stirring for 1h at 90 ℃ by using a magnetic stirrer;
1.2 centrifuging at 3000rpm for 5min, discarding the supernatant, adding 900mL deionized water, adjusting pH to 4.5 with HCl, diluting to 1L, and heating and stirring with a magnetic stirrer at 70 deg.C for 1 h;
1.3 centrifuging for 5min at the rotating speed of 3000rpm, collecting the precipitate, and washing for 3 times by using deionized water;
1.4 centrifuging and collecting the precipitate, and washing with isopropanol for 4 times; centrifuging, collecting the precipitate, and washing with acetone for 2 times;
1.5 collecting the precipitate, naturally drying, and drying in a drying oven for 24h to remove acetone to obtain dextran powder.
Preparation of dextran microsphere-recombinant protein complex
2.1 weighing glucan powder in a centrifuge tube, and washing with deionized water for 3 times;
2.2 adding deionized water into the precipitate obtained in the step 2.1 and performing ultrasonic dispersion for 27min to obtain glucan suspension;
2.3 mixing the glucan suspension with carbodiimide, adjusting the pH to 4.3 by using HCl after complete connection, and stirring for 15 hours at room temperature;
2.4 centrifuging at 5200rpm for 100s, collecting precipitate, washing with deionized water for 3 times;
2.5 resuspending and precipitating with deionized water, adding recombinant protein A/G, mixing uniformly, adjusting the pH value to 4.6 with NaOH, and stirring and incubating at room temperature for 8 h;
2.6 centrifuging, collecting the precipitate, and washing with sterile PBS buffer solution for 3 times;
and 2.7, adding PBS (phosphate buffer solution) containing 0.02 percent of sodium azide for resuspension to obtain the glucan microsphere-recombinant protein compound.
The dextran microsphere-recombinant protein composite can be prepared into an immunoprecipitation reagent based on dextran microspheres.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
Example 2
The embodiment provides a preparation method of an immunoprecipitation reagent based on dextran microspheres, which comprises the following steps:
preparation of yeast glucan:
1.1 adding 100g of high-activity yeast into 1 mass of LNaOH (1M/L), and heating and stirring for 1 hour at 90 ℃ by using a magnetic stirrer;
1.2 centrifuging at 3000rpm for 5min, discarding the supernatant, adding 900mL deionized water, adjusting pH to 4.5 with HCl, diluting to 1L, and heating and stirring with a magnetic stirrer at 70 deg.C for 1 h;
1.3 centrifuging for 5min at the rotating speed of 3000rpm, collecting the precipitate, and washing for 3 times by using deionized water;
1.4 centrifuging and collecting the precipitate, and washing with isopropanol for 4 times; centrifuging, collecting the precipitate, and washing with acetone for 2 times;
1.5 collecting the precipitate, naturally drying, and drying in a drying oven for 24h to remove acetone to obtain dextran powder.
Preparation of dextran microsphere-recombinant protein complex
2.1 weighing glucan powder in a centrifuge tube, and washing with deionized water for 3 times;
2.2 adding the precipitate obtained in the step 2.1 into deionized water and carrying out ultrasonic dispersion for 36min to obtain glucan suspension;
2.3 mixing the glucan suspension with carbodiimide, adjusting the pH to 4.6 by using HCl after complete connection, and stirring and incubating for 20 hours at room temperature;
2.4 centrifuging at 4700rpm for 150s, collecting the precipitate, and washing with deionized water for 3 times;
2.5 resuspending the precipitate with deionized water, adding recombinant protein proteinA A/G-fluorophore (wherein the fluorophore can be selected from one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED), mixing, adjusting pH to 4.3 with NaOH, and incubating at room temperature for 4h under stirring;
2.6 centrifuging, collecting the precipitate, and washing with sterile PBS buffer solution for 3 times;
and 2.7, adding PBS (phosphate buffer solution) containing 0.02 percent of sodium azide for resuspension to obtain the glucan microsphere-recombinant protein compound.
The dextran microsphere-recombinant protein composite can be prepared into an immunoprecipitation reagent based on dextran microspheres.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
Example 3
The embodiment provides a preparation method of an immunoprecipitation reagent based on dextran microspheres, which comprises the following steps:
preparation of yeast glucan:
1.1 adding 100g of high-activity yeast into 1L of NaOH (1M/L), and heating and stirring for 1h at 90 ℃ by using a magnetic stirrer;
1.2 centrifuging at 3000rpm for 5min, discarding the supernatant, adding 900mL deionized water, adjusting pH to 4.5 with HCl, diluting to 1L, and heating and stirring with a magnetic stirrer at 70 deg.C for 1 h;
1.3 centrifuging for 5min at the rotating speed of 3000rpm, collecting the precipitate, and washing for 3 times by using deionized water;
1.4 centrifuging and collecting the precipitate, and washing with isopropanol for 4 times; centrifuging, collecting the precipitate, and washing with acetone for 2 times;
1.5 collecting the precipitate, naturally drying, and drying in a drying oven for 24h to remove acetone to obtain dextran powder.
Preparation of dextran microsphere-recombinant protein complex
2.1 weighing glucan powder in a centrifuge tube, and washing with deionized water for 3 times;
2.2 adding the precipitate obtained in the step 2.1 into deionized water and carrying out ultrasonic dispersion for 30min to obtain a glucan suspension;
2.3 mixing the glucan suspension with carbodiimide, adjusting the pH to 4.5 by using HCl after complete connection, and stirring and incubating for 16h at room temperature;
2.4 centrifuging at 5000rpm for 120s, collecting the precipitate, and washing with deionized water for 3 times;
2.5 resuspending the precipitate with deionized water, adding recombinant protein proteinA A/G-fluorophore (wherein the fluorophore can be selected from one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED), mixing, adjusting pH to 4.5 with NaOH, and incubating at room temperature for 6 h;
2.6 centrifuging, collecting the precipitate, and washing with sterile PBS buffer solution for 3 times;
2.7 adding PBS solution containing 0.02% sodium azide for resuspension to obtain the dextran microsphere-recombinant protein compound, and preparing immunoprecipitate based on dextran microsphere.
The dextran microsphere-recombinant protein composite can be prepared into an immunoprecipitation reagent based on dextran microspheres.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
Example 4
The embodiment provides a preparation method of an immunoprecipitation reagent based on dextran microspheres, which comprises the following steps:
preparation of yeast glucan:
1.1 adding 100g of high-activity yeast into 1L of NaOH (1M/L), and heating and stirring for 1h at 90 ℃ by using a magnetic stirrer;
1.2 centrifuging at 3000rpm for 5min, discarding the supernatant, adding 900mL deionized water, adjusting pH to 4.5 with HCl, diluting to 1L, and heating and stirring with a magnetic stirrer at 70 deg.C for 1 h;
1.3 centrifuging for 5min at the rotating speed of 3000rpm, collecting the precipitate, and washing for 3 times by using deionized water;
1.4 centrifuging and collecting the precipitate, and washing with isopropanol for 4 times; centrifuging, collecting the precipitate, and washing with acetone for 2 times;
1.5 collecting the precipitate, naturally drying, and drying in a drying oven for 24h to remove acetone to obtain dextran powder.
Preparation of dextran microsphere-recombinant protein complex
2.1 weighing glucan powder in a centrifuge tube, and washing with deionized water for 3 times;
2.2 adding the precipitate obtained in the step 2.1 into deionized water and carrying out ultrasonic dispersion for 32min to obtain a glucan suspension;
2.3 mixing the glucan suspension with carbodiimide, adjusting the pH to 4.4 by using HCl after complete connection, and stirring and incubating for 17 hours at room temperature;
2.4 centrifuging at 5100rpm for 140s, collecting the precipitate, washing 3 times with deionized water;
2.5 resuspending the precipitate with deionized water, adding recombinant protein proteinA A/G-fluorophore (wherein the fluorophore can be selected from one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED), mixing, adjusting pH to 4.5 with NaOH, and incubating at room temperature for 8h under stirring;
2.6 centrifuging, collecting the precipitate, and washing with sterile PBS buffer solution for 3 times;
2.7 adding PBS solution containing 0.02% sodium azide for resuspension to obtain the dextran microsphere-recombinant protein compound, and preparing immunoprecipitate based on dextran microsphere.
The dextran microsphere-recombinant protein composite can be prepared into an immunoprecipitation reagent based on dextran microspheres.
The application of the immunoprecipitation reagent based on the dextran microspheres in an immunoprecipitation test.
Examples of the experiments
This experimental example specifically prepares dextran microsphere-recombinant protein complexes and immunoprecipitation reagents based on dextran microspheres with the preparation method of example 3. And carrying out antibody purification experiments and immunoprecipitation, and checking the effect of the immunoprecipitation reagent based on dextran microspheres.
Preparation of dextran microsphere-recombinant protein complex
2.1 weighing 30mg of glucan powder in a centrifuge tube, and washing for 3 times by deionized water;
2.2 adding 6mL of deionized water into the precipitate obtained in the step 2.1, and performing ultrasonic dispersion for 30min to completely disperse the microspheres to obtain a glucan suspension;
2.3 mixing the glucan suspension with 120mg carbodiimide, adjusting the pH to 4.5 by using HCl after complete connection, and stirring and incubating for 16h at room temperature;
2.4 centrifuging at 5000rpm for 120s, collecting the precipitate, and washing with deionized water for 3 times;
2.5 resuspending the precipitate with 15mL of deionized water, adding 1mL of recombinant protein proteinA/G-FITC (2mg/mL), mixing, adjusting the pH value to 4.5 with NaOH, and incubating for 6h at room temperature with stirring;
2.6 centrifuging, collecting the precipitate, and washing with sterile PBS buffer solution for 3 times;
2.7 adding 5mL PBS solution containing 0.02% sodium azide for resuspension to obtain the dextran microsphere-recombinant protein complex.
The dextran microsphere and the recombinant protein are coupled to prepare the dextran microsphere-recombinant protein compound, and the change before and after coupling can be seen from figure 1.
Application of prepared glucan microsphere-recombinant protein complex in antibody purification
3.1 adding 4 ug mouse, rabbit and goat IgG into 800 ul PBS solution to dilute;
3.2 Add 80. mu.L dextran microsphere-recombinant protein complex (10mg/mL) to the tubes containing mouse, rabbit and goat IgG, respectively, incubate 4h at 4 ℃;
3.3 centrifuging at 3000rpm for 5min, collecting the precipitate, and washing 3 times with PBS;
3.4 SDS-PAGE gel electrophoresis detection is carried out on mouse, rabbit and goat IgG samples before purification and mouse, rabbit and goat IgG samples after purification respectively.
The results of SDS-PAGE gel electrophoresis are shown in FIG. 2, and it can be seen that the purified mouse, rabbit and goat IgG samples are adjusted more clearly and the concentration is obviously increased.
Application of glucan microsphere-recombinant protein complex in immunoprecipitation
4.1 culturing HEK293T cells, using lipofectamine 2000 transfection pCMV6-mANKIB1 plasmid (expression of myc-ANKIB1-FLAG fusion protein);
4.2 after transfection for 24h, adding 500. mu.L of RIPA lysate to lyse the cells, lysing for 15min, centrifuging at 12000rpm for 10mn, and collecting the supernatant;
4.3 Add 4. mu.g myc antibody to incubate and 80. mu.L dextran microsphere-recombinant protein complex (10mg/mL) to the supernatant, incubate for 4 h;
4.4 the samples before and after precipitation are respectively subjected to western blot detection for precipitation effect.
The result of western blot detection is shown in FIG. 3, where Input: the lysate supernatant of HEK293T cells transfected by pCMV6-mANKIB1, and the FLAG antibody detection lane; IgG: rabbit IgG pellet, FLAG antibody detection lane, which is a negative control. IP, myc antibody precipitation, and detecting myc-ANKIB1-FLAG fusion protein by using a FLAG antibody. It can be seen that after antibody precipitation, the concentration level is increased and the detection band is clearly visible.
In summary, in the method for preparing an immunoprecipitation reagent based on dextran microspheres provided by the embodiment of the invention, the yeast cells used are baking powder used in daily life, and the price is low. The preparation process of the method is simple, the reagents and equipment used in the process are common in a laboratory, and no high-end precision equipment is needed. And the operation is not needed before the use, the device can be directly used, is simple and quick, and reduces the experiment time. The yeast has uniform size. Therefore, the particle size of the prepared GPs is about 3-4 mu m, and the technology avoids the phenomenon that the traditional stirring and emulsifying preparation of the agarose beads has uneven particle size and poor separation effect caused by uneven pressure. GPs not only acts as a carrier, but is also an FDA approved dietary supplement. The acid-base method is rapid and efficient, can obtain GPs with higher purity, and is suitable for large-scale extraction and purification of glucan. The method is suitable for coupling various target proteins, such as biotin and streptavidin. In addition, the carbodiimide used in the method has better biocompatibility and is often used as a coupling agent in polypeptide and carrier protein or antibody labeling. The experimental reaction condition is mild, the steps are simple, and the influence on the activity of the target protein is small. The method is not only suitable for the conventional immunoprecipitation technology, but also can be applied to the co-immunoprecipitation to research the interaction between proteins, and can be used for purifying antibodies.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A preparation method of an immunoprecipitation reagent based on dextran microspheres is characterized by comprising the following steps:
washing glucan, adding water for dissolving, and performing ultrasonic dispersion to obtain glucan suspension;
mixing the glucan suspension with carbodiimide, adjusting the pH value to 4.3-4.6, performing first incubation, centrifuging and washing to obtain glucan microsphere precipitate;
resuspending the dextran microsphere precipitate, mixing the dextran microsphere precipitate with recombinant protein, adjusting the pH value to 4.3-4.6, performing second incubation, and centrifuging to obtain dextran microsphere-recombinant protein precipitate;
washing the dextran microsphere-recombinant protein precipitate with PBS buffer solution, then resuspending to obtain a dextran microsphere-recombinant protein compound, and preparing the immunoprecipitation reagent based on the dextran microsphere.
2. The method of claim 1, wherein the recombinant protein is one of protein A/G, protein A/G-fluorophore or a secondary antibody.
3. The method of claim 2, wherein the fluorophore is one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, or NED.
4. The method for preparing a dextran microsphere based immunoprecipitation reagent according to claim 1, wherein the time for ultrasonic dispersion is 27-36 min.
5. The method of claim 1, wherein the first incubation is performed with HCl to adjust the pH to 4.3-4.6; the first incubation is at room temperature for 15-20 h.
6. The method of claim 1, wherein the adjusting the pH to 4.3-4.6 and the performing the second incubation are performed with NaOH, and the second incubation is performed at room temperature for 4-8 h.
7. The method for preparing the immunoprecipitation reagent based on dextran microspheres as described in claim 1, wherein the rotation speed of the centrifugation and washing to obtain the dextran microsphere precipitate is 4700-5200rpm for 100-150 s.
8. The method for preparing a dextran microsphere based immunoprecipitation reagent as described in claim 1, wherein said resuspension is performed using PBS buffer containing 0.02% sodium azide after washing said dextran microsphere-recombinant protein precipitate.
9. A dextran microsphere based immunoprecipitation reagent prepared by the method of any one of claims 1 to 8.
10. Use of the dextran microsphere based immunoprecipitation reagent of claim 9 in an immunoprecipitation assay.
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