CN109270064B - Dextran microsphere-based immunoprecipitation reagent and preparation method and application thereof - Google Patents

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 dextran microspheres provided by the present invention uses dextran particles with uniform particle size, which is convenient to obtain materials, simple in the preparation process of microspheres, can be produced on a large scale, and is cheap; The process can rapidly prepare a large number of immunoprecipitation reagents with better experimental effects; the prepared dextran microsphere-based immunoprecipitation reagents are applied in experiments, and the results are accurate and reliable, which has good popularization and application value.

Description

Dextran microsphere-based immunoprecipitation reagent and preparation method and application thereof

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 technique

Immunoprecipitation is a classic method used to study protein-protein, protein-DNA, and protein-RNA interactions in the process of life science research, and it is also an effective method to determine the interaction between target proteins and target molecules under physiological conditions. The principle is to use the specific binding of the antibody to the target molecule, and then add protein A/G that specifically adsorbs the Fc domain of IgG, and the protein A/G is coupled to the surface of the solid phase carrier, and is carried out by centrifugation or magnetic separator. Isolation of immune complexes. Therefore, this method provides technical support for elucidating the mutual regulation network in the life process.

Immunoprecipitation is also one of the indispensable methods for antibody purification. In this method, proteinA/G is covalently coupled to a solid-phase medium, such as Agarose beads or Magnetic beads. When ascites fluid or hybridoma cell supernatant is enriched from the solid-phase medium, the antibody binds to protein A/G. , and then achieve the purpose of enriching antibodies, the method is easy to operate and has high purification efficiency.

Currently, protein A/G mainly uses Agarose beads or Magnetic beads as solid-phase medium. Their preparation process is relatively complicated, the particle size is not uniform, the pressure change during separation is large, which leads to poor separation effect and is expensive.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method of an immunoprecipitation reagent based on dextran microspheres. The preparation method can quickly prepare a solid-phase medium with uniform particle size through a simple preparation process, and the raw materials are cheap.

The second object of the present invention is to provide an immunoprecipitation reagent based on dextran microspheres, which has a uniform particle size, and avoids inconsistency in particle size caused by large pressure changes during separation.

The third object of the present invention is to provide the application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation experiments.

In order to realize the above-mentioned purpose of the present invention, the following technical solutions are adopted:

A preparation method of an immunoprecipitation reagent based on dextran microspheres, comprising the following steps:

Take glucan for washing, add water to dissolve, and ultrasonically disperse to obtain glucan suspension;

Mix the dextran suspension with carbodiimide, adjust the pH to 4.3-4.6 and carry out the first incubation, centrifugation and washing to obtain the dextran microsphere precipitation;

Resuspend the dextran microsphere precipitation and mix it with the recombinant protein, adjust the pH value to 4.3-4.6 and carry out the second incubation and centrifugation to obtain the dextran microsphere-recombinant protein precipitation;

The dextran microsphere-recombinant protein precipitate was washed with PBS buffer and then resuspended to obtain a dextran microsphere-recombinant protein complex, and a dextran microsphere-based immunoprecipitation reagent was prepared.

A dextran microsphere-based immunoprecipitation reagent is prepared by the above-mentioned preparation method of a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

Compared with the prior art, the beneficial effects of the present invention include: the method for preparing the immunoprecipitation reagent based on dextran microspheres provided by the present invention uses dextran particles with uniform particle size, which is convenient to obtain materials and the preparation process of microspheres. It is simple, can be produced on a large scale, and is cheap; through a simple process, a large number of immunoprecipitation reagents with better experimental effects can be prepared quickly; the prepared dextran microsphere-based immunoprecipitation reagents are used in experiments. The results are accurate and reliable, and have good promotion and application value.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 provides the comparison diagram before and after coupling of dextran microspheres and proteinA/G for the embodiment of the present invention;

Fig. 2 is the result diagram of western blot experiment before and after the sample purification that the experimental example of the present invention provides;

FIG. 3 is a graph showing the results of the immunoprecipitation experiment of the dextran microsphere-recombinant protein complex provided by the experimental example of the present invention.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

In order to make the purposes, 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 accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, but not all embodiments. Thus, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

A dextran microsphere-based immunoprecipitation reagent according to the embodiment of the present invention and its preparation method and application will be specifically described below.

A preparation method of an immunoprecipitation reagent based on dextran microspheres, comprising the following steps:

Take glucan for washing, add water to dissolve, and ultrasonically disperse to obtain glucan suspension;

Mix the dextran suspension with carbodiimide, adjust the pH to 4.3-4.6 and carry out the first incubation, centrifugation and washing to obtain the dextran microsphere precipitation;

Resuspend the dextran microsphere precipitation and mix it with the recombinant protein, adjust the pH value to 4.3-4.6 and carry out the second incubation and centrifugation to obtain the dextran microsphere-recombinant protein precipitation;

The dextran microsphere-recombinant protein precipitate was washed with PBS buffer and then resuspended to obtain a dextran microsphere-recombinant protein complex, and a dextran microsphere-based immunoprecipitation reagent was prepared.

The glucan in the experiment can be purchased or homemade; in this experiment, yeast glucan was selected and obtained by homemade.

Further, in a preferred embodiment of the present invention, the recombinant protein package is one of protein A/G, protein A/G-fluorophore or secondary antibody.

Further, in a preferred embodiment of the present invention, the fluorescent group is one of FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 or NED.

Further, in a preferred embodiment of the present invention, the ultrasonic dispersion time is 27-36 min.

Through ultrasonic dispersion, the agglomerated glucan particles can be dispersed to form a uniform glucan solution, which is beneficial to the subsequent experiments.

Further, in a preferred embodiment of the present invention, the pH is adjusted to a value of 4.3-4.6 and HCl is selected for adjustment during the first incubation; the first incubation is incubated at room temperature for 15-20 hours.

Further, in a preferred embodiment of the present invention, the pH value is adjusted to 4.3-4.6 and NaOH is used for the second incubation, and the second incubation is incubated at room temperature for 4-8 hours.

Further, in a preferred embodiment of the present invention, the temperature of the second shaking culture is 20-35°C, and the culture time is 68-75h.

Further, in a preferred embodiment of the present invention, the rotation speed of centrifugation and washing to obtain dextran microsphere precipitation is 4700-5200 rpm, and the time is 100-150 s.

Further, in a preferred embodiment of the present invention, after washing the dextran microsphere-recombinant protein precipitate, select PBS buffer containing 0.02% sodium azide for resuspending.

Re-suspending with PBS buffer containing sodium azide can make the prepared dextran microsphere-based immunoprecipitation reagent stably preserved.

A dextran microsphere-based immunoprecipitation reagent is prepared by the above-mentioned preparation method of a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

The present embodiment provides a preparation method of a dextran microsphere-based immunoprecipitation reagent, comprising the following steps:

Preparation of yeast glucan:

1.1 Add 100g of highly active yeast to 1L of NaOH (1M/L), heat and stir with a magnetic stirrer at 90°C for 1h;

1.2 Centrifuge at 3000 rpm for 5 min, discard the supernatant, add 900 mL of deionized water, adjust the pH to 4.5 with HCl, dilute to 1 L, and heat and stir with a magnetic stirrer at 70 °C for 1 h;

1.3 Centrifuge at 3000rpm for 5min, collect the precipitate, and wash 3 times with deionized water;

1.4 The precipitate was collected by centrifugation and washed 4 times with isopropanol; the precipitate was collected by centrifugation and washed twice with acetone;

1.5 After collecting the precipitate and drying it naturally, it was dried in a drying box for 24 hours to remove acetone to obtain dextran powder.

Preparation of Dextran Microsphere-Recombinant Protein Complex

2.1 Weigh the dextran powder into a centrifuge tube and wash it with deionized water 3 times;

2.2 Add the precipitate in 2.1 to deionized water and ultrasonically disperse for 27min to obtain a dextran suspension;

2.3 Mix the dextran suspension with carbodiimide, after complete connection, adjust the pH to 4.3 with HCl, and stir at room temperature for 15h;

2.4 Centrifuge at 5200rpm for 100s, collect the precipitate, and wash 3 times with deionized water;

2.5 After resuspending the precipitate with deionized water, adding recombinant protein proteinA/G and mixing, adjusting the pH value to 4.6 with NaOH, and incubating at room temperature for 8h;

2.6 The precipitate was collected by centrifugation and washed 3 times with sterile PBS buffer;

2.7 Add 0.02% sodium azide-containing PBS solution to resuspend to obtain a dextran microsphere-recombinant protein complex.

The dextran microsphere-recombinant protein complex can be prepared to obtain a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

Example 2

The present embodiment provides a preparation method of a dextran microsphere-based immunoprecipitation reagent, comprising the following steps:

Preparation of yeast glucan:

1.1 Add 100g of highly active yeast to LNaOH (1M/L) of 1, heat and stir with a magnetic stirrer at 90°C for 1h;

1.2 Centrifuge at 3000 rpm for 5 min, discard the supernatant, add 900 mL of deionized water, adjust the pH to 4.5 with HCl, dilute to 1 L, and heat and stir with a magnetic stirrer at 70 °C for 1 h;

1.3 Centrifuge at 3000rpm for 5min, collect the precipitate, and wash 3 times with deionized water;

1.4 The precipitate was collected by centrifugation and washed 4 times with isopropanol; the precipitate was collected by centrifugation and washed twice with acetone;

1.5 After collecting the precipitate and drying it naturally, it was dried in a drying box for 24 hours to remove acetone to obtain dextran powder.

Preparation of Dextran Microsphere-Recombinant Protein Complex

2.1 Weigh the dextran powder into a centrifuge tube and wash it with deionized water 3 times;

2.2 Add the precipitate in 2.1 to deionized water and ultrasonically disperse it for 36min to obtain a dextran suspension;

2.3 Mix the dextran suspension with carbodiimide, after complete connection, adjust the pH to 4.6 with HCl, and incubate with stirring at room temperature for 20h;

2.4 Centrifuge at 4700rpm for 150s, collect the precipitate, and wash 3 times with deionized water;

2.5 After resuspending the pellet with deionized water, add recombinant protein proteinA/G-fluorophore (wherein the fluorophore can be selected from: FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610 , TEXASRED, RED670 or NED) after mixing, adjust the pH value to 4.3 with NaOH, and incubate at room temperature for 4h;

2.6 The precipitate was collected by centrifugation and washed 3 times with sterile PBS buffer;

2.7 Add 0.02% sodium azide-containing PBS solution to resuspend to obtain a dextran microsphere-recombinant protein complex.

The dextran microsphere-recombinant protein complex can be prepared to obtain a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

Example 3

The present embodiment provides a preparation method of a dextran microsphere-based immunoprecipitation reagent, comprising the following steps:

Preparation of yeast glucan:

1.1 Add 100g of highly active yeast to 1L of NaOH (1M/L), heat and stir with a magnetic stirrer at 90°C for 1h;

1.2 Centrifuge at 3000 rpm for 5 min, discard the supernatant, add 900 mL of deionized water, adjust the pH to 4.5 with HCl, dilute to 1 L, and heat and stir with a magnetic stirrer at 70 °C for 1 h;

1.3 Centrifuge at 3000rpm for 5min, collect the precipitate, and wash 3 times with deionized water;

1.4 The precipitate was collected by centrifugation and washed 4 times with isopropanol; the precipitate was collected by centrifugation and washed twice with acetone;

1.5 After collecting the precipitate and drying it naturally, it was dried in a drying box for 24 hours to remove acetone to obtain dextran powder.

Preparation of dextran microsphere-recombinant protein complex

2.1 Weigh the dextran powder into a centrifuge tube and wash it with deionized water 3 times;

2.2 Add the precipitate in 2.1 to deionized water and ultrasonically disperse for 30min to obtain a dextran suspension;

2.3 Mix the dextran suspension with carbodiimide, after complete connection, adjust the pH to 4.5 with HCl, and incubate with stirring at room temperature for 16h;

2.4 Centrifuge at 5000rpm for 120s, collect the precipitate, and wash 3 times with deionized water;

2.5 After resuspending the pellet with deionized water, add recombinant protein proteinA/G-fluorophore (wherein the fluorophore can be selected from: FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610 , TEXASRED, RED670 or NED) after mixing, adjust the pH value to 4.5 with NaOH, and incubate with stirring at room temperature for 6h;

2.6 The precipitate was collected by centrifugation and washed 3 times with sterile PBS buffer;

2.7 Add 0.02% sodium azide-containing PBS solution for resuspension to obtain a dextran microsphere-recombinant protein complex, and prepare a dextran microsphere-based immunoprecipitation.

The dextran microsphere-recombinant protein complex can be prepared to obtain a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

Example 4

The present embodiment provides a preparation method of a dextran microsphere-based immunoprecipitation reagent, comprising the following steps:

Preparation of yeast glucan:

1.1 Add 100g of highly active yeast to 1L of NaOH (1M/L), heat and stir with a magnetic stirrer at 90°C for 1h;

1.2 Centrifuge at 3000 rpm for 5 min, discard the supernatant, add 900 mL of deionized water, adjust the pH to 4.5 with HCl, dilute to 1 L, and heat and stir with a magnetic stirrer at 70 °C for 1 h;

1.3 Centrifuge at 3000rpm for 5min, collect the precipitate, and wash 3 times with deionized water;

1.4 The precipitate was collected by centrifugation and washed 4 times with isopropanol; the precipitate was collected by centrifugation and washed twice with acetone;

1.5 After collecting the precipitate and drying it naturally, it was dried in a drying box for 24 hours to remove acetone to obtain dextran powder.

Preparation of dextran microsphere-recombinant protein complex

2.1 Weigh the dextran powder into a centrifuge tube and wash it with deionized water 3 times;

2.2 Add the precipitate in 2.1 to deionized water and ultrasonically disperse for 32min to obtain a dextran suspension;

2.3 Mix the dextran suspension with carbodiimide, after complete connection, adjust the pH to 4.4 with HCl, and incubate with stirring at room temperature for 17h;

2.4 Centrifuge at 5100rpm for 140s, collect the precipitate, and wash 3 times with deionized water;

2.5 After resuspending the pellet with deionized water, add recombinant protein proteinA/G-fluorophore (wherein the fluorophore can be selected from: FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610 , TEXASRED, RED670 or NED) after mixing, adjust the pH value to 4.5 with NaOH, and incubate at room temperature for 8h;

2.6 The precipitate was collected by centrifugation and washed 3 times with sterile PBS buffer;

2.7 Add 0.02% sodium azide-containing PBS solution for resuspension to obtain a dextran microsphere-recombinant protein complex, and prepare a dextran microsphere-based immunoprecipitation.

The dextran microsphere-recombinant protein complex can be prepared to obtain a dextran microsphere-based immunoprecipitation reagent.

Application of the above-mentioned dextran microsphere-based immunoprecipitation reagent in immunoprecipitation assay.

Experimental example

In this experimental example, the dextran microsphere-recombinant protein complex and the dextran microsphere-based immunoprecipitation reagent were specifically prepared by the preparation method of Example 3. Antibody purification experiments and immunoprecipitation were carried out to test the effect of immunoprecipitation reagents based on dextran microspheres.

Preparation of dextran microsphere-recombinant protein complex

2.1 Weigh 30 mg of dextran powder into a centrifuge tube and wash with deionized water 3 times;

2.2 Add the precipitation in 2.1 to 6 mL of deionized water and ultrasonically disperse for 30 min to completely disperse the microspheres to obtain a dextran suspension;

2.3 Mix the dextran suspension with 120 mg of carbodiimide, after complete connection, adjust the pH to 4.5 with HCl, and incubate with stirring at room temperature for 16 hours;

2.4 Centrifuge at 5000rpm for 120s, collect the precipitate, and wash 3 times with deionized water;

2.5 After resuspending the pellet with 15 mL of deionized water, add 1 mL of recombinant protein proteinA/G-FITC (2 mg/mL) and mix well, adjust the pH to 4.5 with NaOH, and incubate with stirring at room temperature for 6 hours;

2.6 The precipitate was collected by centrifugation and washed 3 times with sterile PBS buffer;

2.7 Add 5 mL of PBS solution containing 0.02% sodium azide to resuspend to obtain a dextran microsphere-recombinant protein complex.

The dextran microspheres-recombinant protein complex is prepared by coupling the dextran microspheres with the recombinant protein. As can be seen from Figure 1, the changes before and after the coupling.

Application of prepared dextran microsphere-recombinant protein complex in antibody purification

3.1 Add 4 μg mouse, rabbit and goat IgG to 800 μL of PBS solution to dilute;

3.2 Add 80 μL of dextran microspheres-recombinant protein complex (10 mg/mL) to the test tubes containing mouse, rabbit and goat IgG respectively, and incubate at 4°C for 4 hours;

3.3 Centrifuge at 3000rpm for 5min, collect the precipitate, and wash 3 times with PBS;

3.4 The mouse, rabbit and goat IgG samples before purification and the purified mouse, rabbit and goat IgG samples were detected by SDS-PAGE gel electrophoresis respectively.

The results of SDS-PAGE gel electrophoresis are shown in Figure 2. It can be seen that the purified mouse, rabbit and goat IgG samples were adjusted to be clearer and the concentration was significantly increased.

Application of Dextran Microsphere-Recombinant Protein Complex in Immunoprecipitation

4.1 Culture HEK293T cells and use lipofectamine 2000 to transfect pCMV6-mANKIB1 plasmid (expressing myc-ANKIB1-FLAG fusion protein);

4.2 After 24 hours of transfection, add 500 μL of RIPA lysis buffer to lyse the cells, lyse for 15 min, centrifuge at 12000 rpm for 10 min, and collect the supernatant;

4.3 Add 4 μg of myc antibody to the supernatant for incubation and 80 μL of dextran microspheres-recombinant protein complex (10 mg/mL), and incubate for 4 h;

4.4 The pre-precipitation and post-precipitation samples were respectively subjected to western blot to detect the precipitation effect.

The western blot detection results are shown in Figure 3. Input: the lysed supernatant of HEK293T cells transfected with pCMV6-mANKIB1, and FLAG antibody detection lane; IgG: rabbit IgG precipitation, FLAG antibody detection lane, this lane is a negative control. IP: myc antibody precipitation, FLAG antibody detection myc-ANKIB1-FLAG fusion protein. It can be seen that after the antibody precipitation, the concentration surface increases, and the detection band is clearly visible.

To sum up, in the preparation method of the glucan microsphere-based immunoprecipitation reagent provided in the embodiment of the present invention, the yeast cell used is the 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 the laboratory, and no high-end precision equipment is required. And it does not need any operation before use, it can be used directly, it is simple and fast, and the experiment time is reduced. Yeast cells are uniform in size. Therefore, the particle size of the prepared GPs is about 3-4 μm. This technology avoids the phenomenon that the particle size of agarose beads is not uniform in the traditional stirring and emulsification preparation, and the separation effect is poor due to the uneven pressure. GPs can not only be used as a carrier, but also a dietary supplement approved by the FDA. The acid-base method is fast, efficient, and can obtain GPs with high purity, which is suitable for large-scale extraction and purification of glucan. It is suitable for the coupling of various target proteins, such as biotin and streptavidin. In addition, the carbodiimide used in this method has good biocompatibility and is often used as a coupling agent in the labeling of polypeptides and carrier proteins or antibodies. The experimental reaction conditions are mild, the steps are simple, and the activity of the target protein is less affected. It is not only suitable for conventional immunoprecipitation techniques, but also can be applied to co-immunoprecipitation to study the interaction between proteins, and can be used for antibody purification.

The above-described embodiments are some, but not all, embodiments of the present invention. The detailed descriptions of the embodiments of the invention are not intended to limit the scope of the invention as claimed, but are merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims (10)

1. a preparation method based on the immunoprecipitation reagent of dextran microsphere, is characterized in that, comprises the following steps: Take glucan for washing, add water to dissolve, and ultrasonically disperse to obtain glucan suspension; Mixing the dextran suspension with carbodiimide, adjusting the pH value to 4.3-4.6 and performing first incubation, centrifugation and washing to obtain dextran microsphere precipitation; Resuspend the dextran microsphere precipitate and mix with the recombinant protein, adjust the pH value to 4.3-4.6 and carry out the second incubation and centrifugation to obtain the dextran microsphere-recombinant protein precipitate; The dextran microsphere-recombinant protein pellet was washed with PBS buffer and then resuspended to obtain a dextran microsphere-recombinant protein complex, and a dextran microsphere-based immunoprecipitation reagent was prepared. 2. the preparation method of the immunoprecipitation reagent based on dextran microspheres according to claim 1, is characterized in that, described recombinant protein is protein A/G, protein A/G-fluorescent group or in secondary antibody A sort of. 3. The preparation method of dextran microsphere-based immunoprecipitation reagent according to claim 2, wherein the fluorescent group is FITC, FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5 , ROX, RED610, TEXASRED, RED670 or NED. 4 . The method for preparing an immunoprecipitation reagent based on dextran microspheres according to claim 1 , wherein the ultrasonic dispersion time is 27-36 min. 5 . 5. The method for preparing an immunoprecipitation reagent based on dextran microspheres according to claim 1, wherein the pH value is adjusted to 4.3-4.6 and HCl is used for adjustment during the first incubation; 1. Incubation Incubate at room temperature for 15-20h. 6. The method for preparing an immunoprecipitation reagent based on dextran microspheres according to claim 1, characterized in that, adjusting the pH value to 4.3-4.6 and performing the second incubation using NaOH for adjustment, the second Incubation Incubate at room temperature for 4-8h. 7. the preparation method of the immunoprecipitation reagent based on dextran microspheres according to claim 1, is characterized in that, the rotating speed that carries out described centrifugation and washing to obtain described dextran microsphere precipitation is 4700-5200rpm, time for 100-150s. 8. The method for preparing an immunoprecipitation reagent based on dextran microspheres according to claim 1, wherein after washing the dextran microspheres-recombinant protein precipitation, a sodium azide containing 0.02% is selected for use PBS buffer for the resuspension. 9. An immunoprecipitation reagent based on dextran microspheres, wherein the immunoprecipitation reagent based on dextran microspheres is composed of the dextran microsphere-based immunoprecipitation reagent according to any one of claims 1-8. The preparation method of immunoprecipitation reagent is prepared. 10. The application of the dextran microsphere-based immunoprecipitation reagent according to claim 9 in immunoprecipitation assay.

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