BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
产品介绍
阿拉丁生产的UltraBio™羧基磁珠偶联试剂盒，提供高质量的UltraBio™羧基磁珠(UltraBio™ Carboxyl Magnetic Beads)，采用EDC和NHS活化偶联方法，可将含伯氨基的抗体、蛋白、多肽等生物配体共价固定于超顺磁羧基磁珠表面，形成稳定酰胺键。试剂盒配套全套活化、偶联、洗涤与封闭缓冲液，无需自行配制复杂试剂，操作简便，室温即可完成偶联反应。用户只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。
磁珠粒径1.2 μm，为单分散超顺磁微球，表面羧基密度高、非特异性吸附低，磁响应快速，重悬分散性良好。每毫升磁珠悬浊液含约10 mg磁珠，可反应结合不少于0.4 mg IgG抗体，具体的最大结合量和生物分子的分子量大小等相关。偶联产物可直接用于化学发光免疫分析、免疫沉淀(IP/Co-IP)、细胞分选、靶标富集、诊断试剂开发等。
反应原理：EDC/NHS活化体系用于羧基磁珠共价偶联生物配体。第一步，在弱酸性条件下，磁珠表面的羧基(-COOH)先与EDC反应，生成不稳定的O-酰基异脲中间体，该中间体易水解失效，加入NHS后，可转化为稳定性更高的NHS活性酯，连接在磁珠表面。第二步，磁珠表面的NHS酯能够与抗体、蛋白、多肽等配体上的伯氨基(-NH₂)发生亲核取代，脱去NHS，形成稳定的酰胺共价键，将生物配体固定在磁珠表面。偶联完成后，使用含小分子伯胺的封闭液淬灭残留NHS活性酯，消除未反应活性位点，降低非特异性吸附。
产品组分
注:发货时各组分依据储存温度(2-8℃/-20℃)独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。 |
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. 羧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)、羧基(乙酸盐、柠檬酸盐)，若有上述物质，应将待偶联蛋白超滤换液至反应缓冲液中。
5. 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。
6. 在正式实验前，需将试剂恢复至室温。
7. 操作过程中，务必穿戴实验服、一次性手套及防护装备。
8. 所有产品仅限于研究用途。
使用说明
1. 试剂准备
1.1 1 × 活化缓冲液配制：用超纯水将10 × 活化缓冲液稀释10倍，制备1 × 活化缓冲液。
1.2 1 × PBST配制：用超纯水将10 × PBST稀释10倍，制备1 × PBST。
2. 羧基磁珠准备
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 1 × 活化缓冲液重悬磁珠，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。
2.3 重复2.2步骤2次即得到洗涤好的磁珠。
3. 羧基磁珠活化
3.1 EDC溶液配制(20 mg/mL)：称取适量EDC，用1 × 活化缓冲液配制浓度为20 mg/mL的EDC溶液。例如称取10 mg的EDC溶于0.5 mL的1 × 活化缓冲液中，混匀即得EDC溶液。
注意：EDC溶液需现用现配。
3.2 NHS溶液配制(20 mg/mL)：称取适量NHS，用1 × 活化缓冲液配制浓度为20 mg/mL的NHS溶液。例如称取10 mg的NHS溶于0.5 mL的1 × 活化缓冲液中，混匀即得NHS溶液。
注意：NHS溶液需现用现配。
3.3 向洗涤好的磁珠中加入50 μL新鲜配制的EDC溶液，用移液器轻轻重悬磁珠。
3.4 再向3.3磁珠中加入50 μL新鲜配置的NHS溶液，涡旋混匀使磁珠充分悬浮，25℃活化30 min。
注意：活化过程中磁珠需保持悬浮状态，活化完成后需尽快偶联。
4. 羧基磁珠偶联生物分子(以抗体为例)
4.1 磁分离。将磁珠置于磁力架上分离磁珠，移去上清液。
4.2 偶联。加入100 μL(0.5-2.0 mg/mL)抗体溶液，用移液器轻轻吹打重悬磁珠，25℃偶联2 h。
注意：抗体需处于合适的缓冲液中，偶联过程中磁珠需保持悬浮状态。
5. 磁珠封闭
5.1 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。
5.2 用200 μL 反应缓冲液重悬磁珠，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 向磁珠中加入200-500 μL封闭液重悬磁珠，25℃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。
6. 磁珠保存
6.1 封闭结束后，将磁珠置于磁力架上分离，移去上清液。
6.2 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。
6.3 加入100 μL 保存液重悬磁珠，保存于2-8℃。
Introduction
The UltraBio™ Carboxyl Magnetic Bead Conjugation Kit manufactured by Aladdin supplies high-quality UltraBio™ Carboxyl Magnetic Beads (UltraBio™ Carboxyl Magnetic Beads). It adopts the EDC and NHS activation-conjugation method, which can covalently immobilize primary amine-containing biological ligands such as antibodies, proteins and peptides onto the surface of superparamagnetic carboxyl magnetic beads to form stable amide bonds. The kit is equipped with a complete set of activation, conjugation, washing and blocking buffers. There is no need to prepare complicated reagents in-house, and the conjugation reaction can be easily performed at room temperature. Users only need to prepare biomolecules to be conjugated such as antibodies. Using 100 μL of bead suspension per conjugation, this kit supports 50 conjugation reactions in total.
The beads have a particle size of 1.2 μm. These monodisperse superparamagnetic microspheres feature high surface carboxyl density, low non-specific adsorption, fast magnetic response and good re-suspendability. The bead suspension contains approximately 10 mg of magnetic beads per mL, capable of conjugating no less than 0.4 mg of IgG antibody. The maximum binding capacity depends on factors such as the molecular weight of the biomolecule. The conjugated products can be directly applied to chemiluminescent immunoassay, immunoprecipitation (IP/Co-IP), cell sorting, target enrichment, diagnostic reagent development and other applications.
Reaction Principle: The EDC/NHS activation system enables covalent conjugation of biomolecules onto carboxyl magnetic beads. In the first step under weakly acidic conditions, the carboxyl groups (-COOH) on bead surfaces react with EDC to form unstable O-acylisourea intermediates, which are susceptible to hydrolysis. With NHS added, these intermediates are converted into more stable bead-bound NHS active esters. In the second step, NHS esters react with primary amino groups (-NH₂) on ligands such as antibodies, proteins and peptides through nucleophilic substitution. NHS is released and stable covalent amide bonds are formed to immobilize biomolecules on beads. After conjugation, blocking buffer containing small-molecule primary amines quenches residual NHS active esters to eliminate unreacted active sites and reduce non-specific adsorption.
Notes: Each component is individually packaged according to its storage temperature (2-8℃/-20℃) upon shipment, with differentiated temperature-controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf-life is 12 months from the date of manufacture. |
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. Carboxyl 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 including BSA. Buffers must be free of amine-containing compounds (e.g., Tris, glycine) and carboxylate-containing compounds (e.g., acetate, citrate). If such contaminants exist, perform buffer exchange of the protein sample into Reaction Buffer by ultrafiltration.
5. 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.
6. Before formal experimentation, the reagents must be returned to room temperature.
7. During operation, always wear a lab coat, disposable gloves, and protective equipment.
8. All products are for research use only.
Instruction for use
1. Reagent Preparation
1.1 Prepare 1 × Activation Buffer: Dilute the 10 × Activation Buffer 10-fold with ultrapure water to prepare 1 × Activation Buffer.
1.2 Preparation 1 × PBST: Dilute 10 × PBST 10-fold with ultrapure water to prepare 1 × PBST.
2. Carboxyl 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 1 × Activation Buffer by gentle pipetting. Place the tube on a magnetic rack for magnetic separation and remove the supernatant.
2.3 Repeat step 2.2 twice to collect washed magnetic beads.
3. Carboxyl Magnetic Bead Activation
3.1 Prepare EDC solution (20 mg/mL): Weigh appropriate EDC and dissolve in 1 × Activation Buffer to a final concentration of 20 mg/mL. As an example, dissolve 10 mg EDC in 0.5 mL 1 × Activation Buffer and mix well.
Note: The EDC solution must be prepared fresh before each use.
3.2 Prepare NHS solution (20 mg/mL): Weigh appropriate NHS and dissolve in 1 × Activation Buffer to a final concentration of 20 mg/mL. As an example, dissolve 10 mg NHS in 0.5 mL 1 × Activation Buffer and mix well.
Note: The NHS solution must be prepared fresh before each use.
3.3 Add 50 μL of freshly prepared EDC solution to the washed beads, and gently resuspend the beads by pipetting.
3.4 Add an additional 50 μL of freshly prepared NHS solution to the beads from step 3.3. Vortex to fully suspend the beads, then incubate at 25℃ for 30 min for activation.
Note: The beads must remain suspended during activation. Carry out conjugation immediately after activation.
4. Conjugation of Biomolecules to Carboxyl Magnetic Beads (demonstrated with antibody)
4.1 Magnetic separation. Place the beads on a magnetic rack for separation and remove the supernatant.
4.2 Conjugation. Add 100 μL of antibody solution (0.5-2.0 mg/mL). Gently resuspend the beads by pipetting and incubate for 2 h at 25℃.
Note: The antibody must be in a suitable buffer. Keep the beads suspended during conjugation.
5. Bead Blocking
5.1 After conjugation, separate the beads on a magnetic rack. Collect the supernatant to measure the residual antibody concentration and calculate the conjugation capacity.
5.2 Resuspend the beads in 200 μL of Reaction Buffer. Gently pipette to disperse the beads, separate on a magnetic rack, and remove the supernatant. Repeat this procedure twice.
5.3 Add 200-500 μL of blocking buffer to resuspend the beads. Incubate for 2-4 h at 25℃ or overnight at 4℃.
Note: Maintain the beads in suspension throughout the reaction.
6. Bead Storage
6.1 After blocking, separate the beads on a magnetic rack and remove the supernatant.
6.2 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.
6.3 Resuspend the beads in 100 μL of storage buffer and store at 2-8℃.
注:发货时各组分依据储存温度(2-8℃/-20℃)独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。 |
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