Preparation method and application of Myc tag antibody magnetic bead

Abstract

The invention provides a preparation method of Myc tag antibody magnetic beads, and belongs to biomedicine. The preparation method comprises the following steps: washing magnetic beads, adding the washed magnetic beads into a magnetic bead activation buffer solution, activating for 20-60 minutes, coupling the activated magnetic beads with an ultra-filtered Myc tag antibody, adding the magnetically separated magnetic beads into a confining liquid, sealing at 4 DEG C for 2 hours, cleaning, and adding a preserving fluid. And the prepared Myc tag antibody magnetic bead also has relatively good specificity, enrichment capacity and stability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method and application of Myc tag antibody magnetic beads. Background Art

[0002] The Myc tag is a small tag with the sequence of Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu. These 11 amino acids can be expressed as antigen epitopes in different protein frameworks and can still recognize the corresponding antibodies. Carboxyl magnetic beads have the characteristics of superparamagnetism, fast magnetic response, monodispersity and submicron particle size. They can bind to antibodies under the action of special chemical reagents (such as EDC). With the unique three-dimensional surface and high specific surface area of ​​magnetic beads, it can ensure that sufficient antibodies are fixed on the surface of magnetic particles and fully maintain the activity of antibodies.

[0003] Myc tag antibody coupled magnetic beads are an important tool that can achieve rapid purification and separation of target proteins by specifically binding to Myc tag fusion proteins. They are widely used in immunological detection, cell imaging, affinity purification, protein engineering and other fields. However, the Myc tag antibody coupled magnetic beads prepared by the existing method cannot remove the interference of primary amine factors in the antibody buffer, resulting in low titer of the antibody magnetic beads after coupling, and the primary amine factors themselves will also couple with carboxyl magnetic beads to compete with antibodies, further resulting in low titer of antibody magnetic beads, and the inability to quickly and accurately measure the coupling efficiency of antibody magnetic beads. There are also carboxyl groups on the surface of antibodies, and it is impossible to distinguish whether the carboxyl groups come from magnetic beads or antibodies, resulting in false positive results; and there is no accurate coupling time. Since the pH of the antibody preservation solution and the coupling buffer are inconsistent, long-term coupling may cause the antibody to be in an inappropriate pH, affecting the service life of the antibody and resulting in poor stability of the antibody magnetic beads; excessive antibody coupling will also lead to increased costs, and too little antibody coupling will result in low titer of the antibody magnetic beads.

[0004] Therefore, how to simply and accurately detect the efficiency of magnetic bead antibody coupling and eliminate the interference of other primary amine factors is crucial to improving the titer of Myc-tagged magnetic bead antibodies. Summary of the invention

[0005] In view of the defects of the prior art, the present invention eliminates the interference of primary amine factors and designs a method for preparing Myc tag antibody magnetic beads. The prepared Myc tag antibody magnetic beads have good specificity, enrichment ability and stability.

[0006] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: The present invention first uses an ultrafiltration tube to filter the antibody before the antibody is coupled to magnetic beads, and uses the magnetic bead reaction solution to replace the antibody buffer to remove the primary amine factor (such as sodium azide) in the Myc tag antibody buffer; then the supernatant at different time periods after the antibody is coupled is taken out, dripped onto the activated PVDF membrane, and then blocked; because the antibody has light and heavy chains, the corresponding secondary antibody is directly incubated, and then developed, and the residual antibody amount in the supernatant after coupling is observed, and the optimal coupling time and the appropriate antibody coupling amount are determined to improve the detection efficiency of the Myc tag antibody coupled to magnetic beads.

[0007] In one aspect, the present invention provides a method for preparing Myc tag antibody magnetic beads, characterized in that the preparation method comprises the following steps: S1. Wash the magnetic beads and add them to the magnetic bead activation buffer for activation for 20-60 min; S2. coupling the activated magnetic beads with the ultrafiltered Myc tag antibody; S3. Add blocking solution to magnetic separation beads at 4°C for 2 hours, wash and add preservation solution.

[0008] In some embodiments, the magnetic beads are carboxyl magnetic beads.

[0009] In some embodiments, in step S1, the washing solution is a MES buffer with a pH of 5-7.

[0010] In some of the embodiments, in step S1, the magnetic bead activation buffer is EDC and Sulf-NHS, with a concentration of 10-100 mg / mL; the mass ratio of EDC, Sulf-NHS and magnetic beads is 1:1:2.

[0011] In some of the embodiments, in step S2, the Myc tag antibody is first filtered through an ultrafiltration tube, then resuspended with MES buffer and coupled with activated magnetic beads at 4°C for 1-5h; the mass ratio of the Myc tag antibody to the magnetic beads is 1:(100-200).

[0012] In some embodiments, the ultrafiltration tube has a molecular weight cutoff of 3-200 kDa.

[0013] In some embodiments, the blocking solution includes 0.05-2M Tris buffer with a pH of 7.2-7.5, 100-200mM NaCl, 0.2-0.8% (v / v) BSA, 1-2% (v / v) PEG 6000, and 0.1-0.3% (v / v) ProClin 300.

[0014] In some embodiments, the preservation solution is a PBS solution containing 0.01-0.8% (v / v) BSA, 0.02-0.07% (v / v) Tween-20, and 0.02-0.07% (v / v) Proclin300, with a pH of 7.4.

[0015] On the one hand, the present invention also provides Myc tag antibody magnetic beads prepared by the preparation method.

[0016] On the other hand, the present invention provides the use of Myc tag antibody magnetic beads prepared by the preparation method in immunoprecipitation or protein purification.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention prepares a Myc tag antibody magnetic bead, which is ultrafiltered before antibody coupling, which not only removes sodium azide and prevents the interference of primary amine groups on coupling, but also the prepared Myc tag antibody magnetic beads have good specificity and enrichment ability, as well as stability, which is conducive to obtaining more accurate experimental results in the subsequent experiment, and also provides new growth opportunities for the magnetic bead market. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the WB test result of Myc tag antibody magnetic beads without ultrafiltration coupled with different magnetic beads.

[0020] Figure 2 This is a graph showing the WB test results of ultrafiltered Myc tag antibody magnetic beads coupled with different magnetic beads.

[0021] Figure 3 This is the WB test result of 5 and 10 μg ultrafiltered Myc tag antibody coupled to magnetic beads.

[0022] Figure 4 This is the WB test result of Myc tag antibody coupled to magnetic beads with different coupling times.

[0023] Figure 5 This is the effectiveness test result of different Myc tag antibody-coupled magnetic beads. In the figure, the size of the non-target band protein is about 25 kD, and the size of the target band is about 60 kD.

[0024] Figure 6 This is a graph showing the stability test results of ultrafiltered Myc tag antibody magnetic beads No. 1.

[0025] Figure 7 This is a graph showing the stability test results of ultrafiltered Myc tag antibody magnetic beads No. 2.

[0026] Figure 8 This is a graph showing the effectiveness test results of Myc tag antibody coupled to magnetic beads at different coupling times. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0029] In the present invention, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] MES: 2-morpholineethanesulfonic acid; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); Sulfo-NHS: N-Hydroxysulfosuccinimide sodium salt; pH: hydrogen ion concentration, hydrogen ion concentration index, used to indicate the acidity or alkalinity of a solution; BSA: Bovine serum albumin; PBS: phosphate buffered saline (PBS), phosphate buffered saline; Proclin 300: Liquid biopreservative; Tween 20: surfactant; PVDF: polyvinylidene fluoride.

[0031] The magnetic beads used in the present invention are carboxyl magnetic beads, which are produced by Guangzhou Yixin Biotechnology Co., Ltd. The particle size of No. 1 carboxyl magnetic beads is 1 μm, and the concentration is 25 mg / mL; the particle size of No. 2 carboxyl magnetic beads is 3 μm, and the concentration is 25 mg / mL; the particle size of No. 3 carboxyl magnetic beads is 1 μm, and the concentration is 10 mg / mL; the particle size of No. 4 carboxyl magnetic beads is 3 μm, and the concentration is 30 mg / mL; the particle size of No. 5 carboxyl magnetic beads is 3 μm, and the concentration is 30 mg / mL.

[0032] The EDC used in the present invention was purchased from Sinopharm Chemical Reagent Co., Ltd. with a product number of XW70841192.

[0033] The Sulfo-NHS used in the present invention was purchased from the Exploration Platform with a product number of 01005378.

[0034] The test of the amount of magnetic bead coupling of the labeled antibody is different from the traditional BSA method and fluorescence method. The BSA method has a large error due to the low concentration of the antibody measured; although the fluorescence method can measure more accurately, it is cumbersome to operate and has a high cost, and is not suitable for large-scale testing. At the same time, there are a large number of carboxyl groups on the surface of the protein coupled to the magnetic beads, and the fluorescent dyes with amino active groups cannot achieve directional coupling. The unreacted binding sites on the surface of the magnetic beads will bind to the coupled proteins, and the fluorescent substances will undergo fluorescence quenching, resulting in inaccurate test results. In addition, the antibody preservation solution also contains factors containing primary amine groups such as BSA and sodium azide, resulting in false positive results. Therefore, the present invention uses WB (Western Blot) to test the amount of magnetic bead coupling of the labeled antibody.

[0035] Example 1 5 μg of unfiltered Myc tag antibody was used to couple 1 mg of magnetic beads No. 1, 2, 3, 4, and 5, respectively.

[0036] 1. Activation of Carboxyl Magnetic Beads 1. Take 1 mg of magnetic beads of each type and wash them with 200 μL 50 mM MES magnetic bead reaction solution; 2. Use the magnetic bead reaction solution to prepare 50 mg / mL EDC and Sulf-NHS activation buffer; 3. Add activation buffer to the magnetic beads, incubate at room temperature for 30 min, and then separate them by magnetic separation. Then add 200 μL 50 mM MES coupling buffer to wash.

[0037] 2. Carboxyl magnetic beads antibody coupling 1. Add antibodies; 2. Incubate at 4°C for 2 hours, take the supernatant after coupling and store it at 4°C; 3. Antibody magnetic bead sealing and storage 1. Magnetic separation beads; 2. Add 200 μL magnetic bead blocking solution (pH 7.4, 0.1M Tris buffer, 150mM NaCl, 0.5% BSA, 2% PEG 6000, 0.2% ProClin 300) 3. Block at 4°C for 2h; 4. Magnetic separation, wash once with 200 μL blocking solution; 5. Add 200 μL of preservation solution (PBS solution containing 0.5% BSA, 0.05% Tween 20, 0.05% ProClin 300, pH=7.4).

[0038] 4. WB (Western Blot) test label antibody magnetic bead coupling amount 1. Take out the PVDF membrane and activate it with methanol for 1 min; 2. Remove methanol and add pure water to wash for 15 min; 3. After air drying at room temperature, add the supernatant after coupling, positive control, and blank control onto the membrane, and then block with 5% BSA on a shaker at room temperature for 1 hour; 4. Remove the blocking solution (5% skim milk powder), add the mouse secondary antibody of the corresponding species, and incubate on a shaker at room temperature for 1 hour; 5. Remove the secondary antibody and wash four times with TBST, 5 min each time; 6. Add developer and expose using WB exposure meter.

[0039] The results are as follows Figure 1 As shown in the figure, magnetic beads No. 1 can completely couple with 5 μg of Myc tag antibody that has not been ultrafiltered, while magnetic beads No. 2, 3, 4, and 5 can hardly couple with the antibody.

[0040] Example 2 5 μg of ultrafiltered Myc tag antibody was coupled to 1 mg of magnetic beads No. 1, 2, 3, 4, or 5.

[0041] 1. Activation of Carboxyl Magnetic Beads 1. Take 1 mg of magnetic beads and wash them with 200 μL 50 mM MES magnetic bead reaction solution; 2. Use the magnetic bead reaction solution to prepare 50 mg / mL EDC and Sulf-NHS activation buffer; 3. Add activation buffer to the magnetic beads, incubate at room temperature for 30 min, then separate magnetically, and add 200 μL 50 mM MES coupling buffer to wash.

[0042] 2. Carboxyl magnetic beads antibody coupling 1. Add the antibody to an ultrafiltration tube (molecular weight cutoff 100 kDa) and centrifuge at 10,000 × g for 10 min. 2. Resuspend with coupling buffer and add to the activated magnetic beads; 3. Incubate at 4°C for 2 h, take the supernatant after coupling and store it at 4°C; 3. Antibody magnetic bead sealing and storage 1. Magnetic separation beads; 2. Add 200 μL magnetic bead blocking solution (pH=7.4 and 0.1M Tris buffer, 150mM NaCl, 0.5% BSA, 0.2% ProClin 300) 3. Block at 4°C for 2h; 4. Magnetic separation, wash once with 200 μL blocking solution; 5. Add 200 μL of preservation solution (PBS solution containing 0.5% BSA, 0.05% Tween 20, 0.05% ProClin 300, pH=7.4).

[0043] 4. WB test: The coupling amount of label antibody magnetic beads is the same as that in Example 1.

[0044] The results are as follows Figure 2 As shown, after ultrafiltration of the antibody, all magnetic beads can be coupled to the antibody.

[0045] Example 3 5 and 10 μg of ultrafiltered Myc tag antibody were coupled to 1 mg of magnetic beads to analyze the upper limit of magnetic bead-coupled antibody.

[0046] 1. Activation of Carboxyl Magnetic Beads 1. Take 1 mg of magnetic beads and wash them with 200 μL 50 mM MES magnetic bead reaction solution; 2. Use the magnetic bead reaction solution to prepare 50 mg / mL EDC and Sulf-NHS activation buffer; 3. Add activation buffer to the magnetic beads, incubate at room temperature for 30 min, then separate by magnetic separation, and add 200 μL 50 mM MES coupling buffer to wash.

[0047] 2. Carboxyl magnetic beads antibody coupling 1. Add the antibody to an ultrafiltration tube (molecular weight cutoff 100 kDa) and centrifuge at 10,000 × g for 10 min. 2. Resuspend with coupling buffer and add to the activated magnetic beads; 3. Incubate at 4°C for 2 h, take the supernatant after coupling and store it at 4°C; 3. Antibody magnetic beads sealed and stored 1. Magnetic separation beads; 2. Add 200 μL magnetic bead blocking solution (pH=7.4 and 0.1M Tris buffer, 150mM NaCl, 0.5% BSA, 0.2% ProClin 300); 3. Block at 4°C for 2h; 4. Magnetic separation, wash once with 200 μL blocking solution; 5. Add 200 μL of preservation solution (PBS solution containing 0.5% BSA, 0.05% Tween 20, 0.05% ProClin 300, pH=7.4).

[0048] 4. WB test: The coupling amount of label antibody magnetic beads is the same as that in Example 1.

[0049] The results are as follows Figure 3 As shown, after ultrafiltration of the Myc tag antibody, the magnetic beads coupled with 10 μg of antibody will reach saturation.

[0050] Example 4 5 μg of ultrafiltered Myc tag antibody was coupled to 1 mg of magnetic beads, and the shortest coupling time of Myc tag antibody magnetic beads was analyzed.

[0051] 1. Activation of Carboxyl Magnetic Beads 1. Take 1 mg of magnetic beads and wash them with 200 μL 50 mM MES magnetic bead reaction solution; 2. Use the magnetic bead reaction solution to prepare 50 mg / mL EDC and Sulf-NHS activation buffer; 3. Add activation buffer to the magnetic beads, incubate at room temperature for 30 min, then separate magnetically, and add 200 μL 50 mM MES coupling buffer to wash.

[0052] 2. Carboxyl magnetic beads antibody coupling 1. Add the antibody to the ultrafiltration tube and centrifuge at 10,000 × g for 10 min; 2. Resuspend with coupling buffer and add to the activated magnetic beads; Incubate at 3, 4 °C for 1, 2, 3, and 4 h, and store the supernatant after coupling at 4 °C; 3. Antibody magnetic bead sealing and storage 1. Magnetic separation beads; 2. Add 200 μL magnetic bead blocking solution (pH=7.4 and 0.1M Tris buffer, 150mM NaCl, 0.5% BSA, 0.2% ProClin 300) 3. Block at 4°C for 2h; 4. Magnetic separation, wash once with 200 μL blocking solution; 5. Add 200 μL of preservation solution (PBS solution with pH=7.4 containing 0.5% BSA, 0.05% Tween 20, and 0.05% ProClin 300).

[0053] 4. WB test: The coupling amount of label antibody magnetic beads is the same as that in Example 1.

[0054] The results are as follows Figure 4 As shown, the shortest antibody magnetic bead coupling time is 2h.

[0055] Example 5 1. Test the effectiveness of Myc tag conjugated to different antibody beads Test magnetic beads: Myc tag antibody magnetic beads No. 1 and No. 2 coupled in Example 2, and commercial Myc tag antibody magnetic beads (the storage solution contains sodium azide).

[0056] Test method: Use cell lysate overexpressing Myc-tagged protein to incubate 20 μL of prepared Myc-tagged antibody magnetic beads and other commercial Myc-tagged antibody magnetic beads. After incubation, wash with IP lysis buffer, wash with protein loading buffer, and then run the SDS-PAGE gel for testing.

[0057] The results are as follows Figure 5 As shown, compared with the commercial Myc tag antibody magnetic beads, the Myc tag antibody magnetic beads No. 1 and No. 2 of the present invention have better specificity and target protein enrichment ability.

[0058] 2. Stability Test Test magnetic beads: Myc tag antibody magnetic beads No. 1 and No. 2 coupled in Example 2.

[0059] The antibody magnetic beads were stored at 37°C for accelerated destruction, and the magnetic beads were incubated with cell lysates (containing Myc-tagged fusion proteins) at 3, 6, and 9 days, respectively, and detected by SDS-PAGE after washing and elution.

[0060] The results are as follows Figure 6 , 7 As shown, Myc tag antibody magnetic beads No. 1 and No. 2 still have good effects after 9 days of accelerated destruction.

[0061] Example 6 Testing the effect of Myc tag antibody magnetic bead coupling time Test magnetic beads: Myc tag antibody magnetic beads coupled for 2h and 4h in Example 4.

[0062] Test method: Incubate 20 μL of two prepared Myc tag antibody magnetic beads with cell lysate that overexpresses Myc tag protein. After incubation, wash with IP lysis buffer, wash with protein loading buffer, and then run the beads on SDS-PAGE.

[0063] The results are as follows Figure 8 As shown, Myc tag antibody magnetic beads coupled for 2h and 4h can completely enrich the Myc tag fusion protein in the cell lysate.

[0064] In summary, the method for preparing Myc tag antibody magnetic beads of the present invention removes sodium azide to prevent the interference of primary amine groups on coupling, and designs a method for detecting the titer of antibody magnetic bead coupling. By using a method similar to WB to detect the supernatant after antibody coupling, it is reflected whether the antibody is completely coupled to the magnetic beads. Compared with the traditional BSA method and fluorescence method, the result is more reliable, the operation is simple, the cost is low, the antibody magnetic bead coupling time and the saturation amount required for antibody coupling can be effectively tested, the antibody cost and time can be effectively saved, and the antibody is prevented from being in the coupling buffer for a long time, which affects the titer of the antibody.

[0065] Meanwhile, the Myc tag antibody magnetic beads developed by the present invention have good effects and stability, and still have a good effect of enriching the target protein after 9 days of accelerated destruction, so the effect is not affected even if placed at 4°C for about 18 months.

[0066] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing Myc tag antibody magnetic beads, characterized in that: The preparation method comprises the following steps: S1. Wash the magnetic beads and add them to the magnetic bead activation buffer for activation for 20-60 min; S2. coupling the activated magnetic beads with the ultrafiltered Myc tag antibody; S3. Add blocking solution to magnetic separation beads at 4°C for 2 hours, wash and add preservation solution.

2. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: The magnetic beads are carboxyl magnetic beads.

3. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: In step S1, the washing solution is a MES buffer solution with a pH of 5-7.

4. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: In step S1, the magnetic bead activation buffer is EDC and Sulf-NHS, with a concentration of 10-100 mg / mL; the mass ratio of EDC, Sulf-NHS and magnetic beads is 1:1:

2.

5. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: In step S2, the Myc tag antibody is first filtered through an ultrafiltration tube, and then resuspended with MES buffer and coupled with activated magnetic beads at 4°C for 1-5 hours; the mass ratio of the Myc tag antibody to the magnetic beads is 1: (100-200).

6. The method for preparing Myc tag antibody magnetic beads according to claim 5, characterized in that: The molecular weight cut-off of the ultrafiltration tube is 3-200 kDa.

7. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: The blocking solution includes 0.05-2M Tris buffer with a pH of 7.2-7.5, 100-200mM NaCl, 0.2-0.8% (v / v) BSA, 1-2% (v / v) PEG6000, and 0.1-0.3% (v / v) ProClin 300.

8. The method for preparing Myc tag antibody magnetic beads according to claim 1, characterized in that: The storage solution is a PBS solution containing 0.01-0.8% (v / v) BSA, 0.02-0.07% (v / v) Tween-20, and 0.02-0.07% (v / v) Proclin 300, with a pH of 7.

4.

9. Myc tag antibody magnetic beads prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of Myc tag antibody magnetic beads prepared according to the preparation method according to any one of claims 1 to 8 in immunoprecipitation or protein purification.

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