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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
阿拉丁生产的UltraBio™甲苯磺酰基磁珠偶联试剂盒，提供高质量UltraBio™甲苯磺酰基活化磁珠(UltraBio™ Tosyl Magnetic Beads)。试剂盒配套完整的偶联、洗涤、封闭与保存缓冲液，无需自行配制复杂试剂，操作简便。用户仅需自备抗体等待偶联的生物分子。按每次使用100 μL磁珠悬液进行偶联，本试剂盒可完成50次偶联反应。
甲苯磺酰基磁珠粒径1.2 μm，磁珠表面带有甲苯磺酰基功能基团，无需额外活化，在温和碱性条件下即可直接和多种生物配体（抗体、活性蛋白、多肽等）上的伯氨基或巯基发生共价偶联反应，分别形成结构稳定、不易降解的二级胺键或硫醚键。甲苯磺酰基磁珠偶联工艺简单、易于放大，是生物大分子固定化的理想载体。每毫升磁珠悬液约含10 mg磁珠，可偶联不少于1 mg IgG抗体；实际最大结合容量取决于生物分子分子量、空间构象等因素。偶联产物可直接用于化学发光免疫分析、免疫沉淀(IP/Co-IP)、靶标富集、诊断试剂开发等应用。
反应原理：甲苯磺酰基磁珠表面呈疏水性，蛋白首先依靠疏水相互作用被动吸附至磁珠表面，随后磁珠表面的甲苯磺酰基与配体上的伯氨基或巯基发生亲核取代反应，形成稳定的二级胺键或硫醚键，偶联完成后的磁珠亲水性显著提高。体系中加入硫酸铵可借助盐析效应促进蛋白在磁珠表面富集，提升偶联效率。共价键生成速率慢于物理吸附，通常需要 37 ℃孵育过夜保障充分偶联。甲苯磺酰基本身不易水解，未反应的残留甲苯磺酰基必须通过BSA、乙醇胺或Tris等含氨基小分子封闭淬灭，消除活性位点，降低非特异性吸附。
产品组分
| U1524128 | 组分 | 物理外观 | 50 reactions | 储存温度 | 每次反应用量 |
| U1524128A | 甲苯磺酰基化磁珠(1.2μm) | 棕黑色混悬液 | 5 mL | 2-8℃，禁止冷冻 | 每次反应需使用100μL |
| U1524128B | 反应缓冲液A | 液体 | 100 mL | 2-8℃ | 请按说明使用 |
| U1524128C | 反应缓冲液B | 液体 | 100 mL | 2-8℃ | 请按说明使用 |
| U1524128D | 缓冲液C | 液体 | 2 mL | 2-8℃ | 请按说明使用 |
| U1524128E | 10×PBST | 液体 | 10 mL | 2-8℃ | 请按说明配制 |
| U1524128F | 封闭液 | 液体 | 25 mL | 2-8℃ | 请按说明使用 |
| U1524128G | 保存液 | 液体 | 5 mL | 2-8℃ | 每次反应需使用100μL |
其他所需试剂
1. 待偶联的抗体等生物分子。
2. 磁力架。
3. 样品混匀仪。
重要须知
1. 磁珠需保存在2-8℃，冷冻、干燥、高速离心均会造成磁珠不可逆团聚、丧失结合活性。
2. 请勿长时间将磁珠置于磁场中，否则可能会引起磁珠聚团。
3. 氨基磁珠使用前要适当充分重悬，即颠倒若干次使磁珠混合均匀，充分震荡或超声使磁珠呈均匀的悬浮状态。
4. 偶联所用的蛋白不能含有BSA之类蛋白类稳定剂，缓冲液也不能含有氨基(Tris、甘氨酸等)，这些物质会竞争活性位点，影响偶联效率。
5. 建议将待偶联蛋白超滤换液至反应缓冲液A或反应缓冲液B中。
6. 共价偶联过程会伴随一定非特异性吸附，可通过后续洗涤步骤降低；非吸附程度随实验体系变化，需根据需求调整洗涤方案；高盐(1 M NaCl)、弱酸碱、适度升温、延长洗涤时间均可减少非共价结合蛋白。
7. 在正式实验前，需将试剂恢复至室温。
8. 操作过程中，务必穿戴实验服、一次性手套及防护装备。
9. 所有产品仅限于研究用途。
使用说明
1. 试剂准备
1.1 1 × PBST配制：用超纯水将10 × PBST稀释10倍，制备1 × PBST。
2. 甲苯磺酰基磁珠准备
2.1 将磁珠充分重悬，移取100 μL(含1 mg磁珠)至离心管中，置于磁力架上，磁吸分离磁珠，移去上清液。
2.2 用500 μL 反应缓冲液A或反应缓冲液B重悬磁珠，用移液器轻轻吹打磁珠，置于磁力架上分离磁珠，移去上清液。
注意：更推荐反应缓冲液A作为偶联缓冲液，建议优先考虑使用；对pH敏感的蛋白建议使用反应缓冲液B重悬磁珠。反应缓冲液A的pH值约为9.5，反应缓冲液B的pH值约为7.3-7.6。
2.3 重复2.2步骤2次即得到洗涤好的磁珠。
3. 甲苯磺酰基磁珠偶联生物分子(以抗体为例)
3.1 偶联。向洗涤好的磁珠中加入66.7 μL(1.5-4.5 mg/mL)抗体溶液，用移液器轻轻吹打重悬磁珠。
注意：抗体需处于合适的缓冲液中，按2.2注意事项选择合适的缓冲液。
3.2 向3.1磁珠反应液中加入33.3 μL Buffer C，涡旋或者吹打充分混匀。37℃偶联12-18 h，或者25℃偶联20 h以上。
注意：偶联过程中磁珠需保持悬浮状态。
4. 磁珠封闭
4.1 偶联结束后，将磁珠置于磁力架上分离，收集上清液，测试偶联后上清液浓度，计算偶联量。
4.2 向磁珠中加入200-500 μL封闭液重悬磁珠，37℃反应2-4 h或者4℃过夜反应。
注意：反应过程中磁珠需保持悬浮状态。
5. 磁珠保存
5.1 封闭结束后，将磁珠置于磁力架上分离，移去上清液。
5.2 用500 μL 1 × PBST重悬磁珠，用移液器轻轻吹打磁珠，置于磁力架上分离磁珠，移去上清液。重复操作2次。
5.3 加入100 μL 保存液重悬磁珠，保存于2-8℃。
UltraBio™ Tosyl Magnetic Bead Conjugation Kit manufactured by Aladdin supplies high-quality UltraBio™ Tosyl Magnetic Beads. The kit is equipped with a complete set of coupling, washing, blocking and storage buffers. There is no need to prepare complicated reagents in-house, and the coupling reaction can be easily performed under mildly alkaline conditions. Users only need to prepare biomolecules to be conjugated such as antibodies. Using 100 μL of bead suspension per coupling, this kit supports 50 coupling reactions in total.
The beads have a particle size of 1.2 μm. These monodisperse superparamagnetic microspheres feature tosyl functional groups on the surface, require no additional activation, and can directly undergo covalent coupling with primary amines or thiols on a variety of biological ligands (antibodies, active proteins, peptides, etc.) under mildly alkaline conditions to form stable, hydrolysis-resistant secondary amine linkages or thioether bonds, respectively. The tosyl magnetic bead coupling process is simple and scalable, making them an ideal carrier for biomacromolecule immobilization. The bead suspension contains approximately 10 mg of magnetic beads per mL, capable of conjugating no less than 1 mg of IgG antibody. The maximum binding capacity depends on factors such as the molecular weight and spatial conformation of the biomolecule. The conjugated products can be directly applied to chemiluminescent immunoassay, immunoprecipitation (IP/Co-IP), target enrichment, diagnostic reagent development and other applications.
Reaction Principle: Tosyl magnetic beads are hydrophobic. Proteins are first passively adsorbed onto the bead surface via hydrophobic interactions. The surface tosyl groups then undergo nucleophilic substitution with primary amines or thiols on the ligand, forming stable secondary amine linkages or thioether bonds. Covalent conjugation renders the beads substantially more hydrophilic. Inclusion of ammonium sulfate promotes protein enrichment on the beads through salting-out effects and improves conjugation efficiency. Covalent bond formation proceeds more slowly than physical adsorption; overnight incubation at 37 °C is typically used to achieve sufficient coupling. Tosyl groups themselves are relatively resistant to hydrolysis. Unreacted residual tosyl groups must be quenched and capped using amine-containing small molecules such as BSA, ethanolamine or Tris to eliminate reactive sites and reduce non-specific adsorption.
Contents and storage
| U1524128 | Component | Appearance | 50 reactions | Storage | Quantity Per reaction |
| U1524128A | Tosyl Magnetic Beads(1.2μm) | Brownish-black suspension | 5 mL | 2-8℃. Do not freeze. | 100 μL for 1 reaction |
| U1524128B | Reaction Buffer A | liquid | 100 mL | 2-8℃. | Use according to instructions |
| U1524128C | Reaction Buffer B | liquid | 100 mL | 2-8℃. | Use according to instructions |
| U1524128D | Buffer C | liquid | 2 mL | 2-8℃. | Use according to instructions |
| U1524128E | 10×PBST | liquid | 10 mL | 2-8℃. | Prepare according to instructions |
| U1524128F | Blocking Solution | liquid | 25 mL | 2-8℃. | Use according to instructions |
| U1524128G | Storage Solution | liquid | 5 mL | 2-8℃. | 100 μL for 1 reaction |
Required materials not supplied
1. Biomolecules to be conjugated, such as antibodies.
2. Magnetic Rack.
3. Sample Mixer.
Important
1. Store magnetic beads at 2-8 °C. Freezing, lyophilization, and high-speed centrifugation cause irreversible bead aggregation and loss of binding activity.
2. Do not expose magnetic beads to a magnetic field for prolonged periods, as this may induce bead aggregation.
3. Tosyl magnetic beads should be adequately resuspended before use. Invert the vial several times to homogenize the beads, followed by gentle vortexing or sonication to obtain a homogeneous suspension.
4. Proteins intended for conjugation should not contain protein stabilizers such as BSA. Buffers must be free of amine-containing compounds (e.g., Tris, glycine). These substances compete with the target ligand for reactive sites and reduce coupling efficiency.
5. It is recommended that the protein sample for conjugation be buffer-exchanged into Reaction Buffer A or Reaction Buffer B by ultrafiltration.
6. Covalent conjugation is accompanied by a certain level of non-specific adsorption, which can be reduced by subsequent washing steps. The degree of non-specific adsorption varies with the assay system; the washing protocol should be optimized according to experimental requirements. High salt (1 M NaCl), mild acid/alkaline conditions, moderate elevation of temperature, and extended washing duration can reduce non-covalently bound protein.
7. Before formal experimentation, the reagents must be returned to room temperature.
8. During operation, always wear a lab coat, disposable gloves, and protective equipment.
9. All products are for research use only.
Instruction for use
1. Reagent Preparation
1.1 Preparation 1 × PBST: Dilute 10 × PBST 10-fold with ultrapure water to prepare 1 × PBST.
2. Tosyl Magnetic Bead Preparation
2.1 Resuspend the magnetic beads completely. Transfer 100 μL (containing 1 mg magnetic beads) to a centrifuge tube. Perform magnetic separation on a magnetic rack and remove the supernatant.
2.2 Resuspend the beads in 500 μL of Reaction Buffer A or Reaction Buffer B by gentle pipetting. Place the tube on a magnetic rack for magnetic separation and remove the supernatant.
Note: Reaction Buffer A is the preferred conjugation buffer. For pH‑sensitive proteins, use Reaction Buffer B for bead resuspension. Reaction Buffer A has a pH of approximately 9.5, while Reaction Buffer B ranges from pH 7.3 to 7.6.
2.3 Repeat step 2.2 twice to collect washed magnetic beads.
3. Conjugation of Biomolecules to Tosyl Magnetic Beads (demonstrated with antibody)
3.1 Conjugation. Add 66.7 μL of antibody solution (1.5-4.5 mg/mL) to the washed magnetic beads, and gently resuspend the beads by pipetting.
Note: Ensure the antibody is in an appropriate buffer, per the Note in Section 2.2.
3.2 Add 33.3 μL of Buffer C to the bead suspension from step 3.1. Mix thoroughly by vortexing or pipetting. Incubate for 12-18 h at 37 °C, or for a minimum of 20 h at 25 °C.
Note: Keep the beads suspended during conjugation.
4. Bead Blocking
4.1 After conjugation, separate the beads on a magnetic rack. Collect the supernatant to measure the residual antibody concentration and calculate the conjugation capacity.
4.2 Add 200-500 μL of blocking buffer to resuspend the beads. Incubate for 2-4 h at 37℃ or overnight at 4℃.
Note: Maintain the beads in suspension throughout the reaction.
5. Bead Storage
5.1 After blocking, separate the beads on a magnetic rack and remove the supernatant.
5.2 Resuspend the beads in 500 μL of 1 × PBST. Gently pipette to disperse the beads, separate on a magnetic rack, and remove the supernatant. Repeat this procedure twice.
5.3 Resuspend the beads in 100 μL of storage buffer and store at 2-8℃.
| U1524128 | 组分 | 物理外观 | 50 reactions | 储存温度 | 每次反应用量 |
| U1524128A | 甲苯磺酰基化磁珠(1.2μm) | 棕黑色混悬液 | 5 mL | 2-8℃，禁止冷冻 | 每次反应需使用100μL |
| U1524128B | 反应缓冲液A | 液体 | 100 mL | 2-8℃. | 请按说明使用 |
| U1524128C | 反应缓冲液B | 液体 | 100 mL | 2-8℃. | 请按说明使用 |
| U1524128D | 缓冲液C | 液体 | 2 mL | 2-8℃. | 请按说明使用 |
| U1524128E | 10×PBST | 液体 | 10 mL | 2-8℃. | 请按说明配制 |
| U1524128F | 封闭液 | 液体 | 25 mL | 2-8℃. | 请按说明使用 |
| U1524128G | 保存液 | 液体 | 5 mL | 2-8℃. | 每次反应需使用100μL |
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