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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™氨基磁珠偶联试剂盒，提供高质量的UltraBio™氨基磁珠(UltraBio™ Amine Magnetic Beads)，采用经典戊二醛交联活化方案，可将带有伯氨基的抗体、蛋白、多肽等生物配体共价固定于磁珠表面。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。用户只需自备戊二醛以及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，表面氨基密度高、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1 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。戊二醛交联条件温和，对蛋白活性友好，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：戊二醛属于同双功能交联剂，其在水溶液中可发生自聚合形成多聚醛结构；多聚戊二醛一端的醛基与氨基磁珠表面的氨基发生反应，另一端的醛基可进一步与抗体、蛋白、多肽等配体上的伯氨基发生交联，形成稳定的共价连接。偶联结束后通过封闭处理淬灭磁珠表面残留的活性醛基，降低磁珠的非特异性吸附。
产品组分
| U1508966 | 组分 | 物理外观 | 50 reactions | 储存温度 | 每次反应用量 |
| U1508966A | 氨基磁珠(1.2μm) | 棕黑色混悬液 | 5 mL | 2-8℃，禁止冷冻 | 每次反应需使用100μL |
| U1508966B | 10×洗涤液 | 液体 | 30 mL | 2-8℃ | 请按说明配制 |
| U1508966C | 反应缓冲液 | 液体 | 10 mL | 2-8℃ | 请按说明使用 |
| U1508966D | 10×PBST | 液体 | 10 mL | 2-8℃ | 请按说明配制 |
| U1508966E | 封闭液 | 液体 | 25 mL | 2-8℃ | 请按说明使用 |
| U1508966F | 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. 戊二醛溶液。
3. 磁力架。
4. 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. 戊二醛具有刺激性、挥发性，操作需在通风橱内进行，佩戴手套、护目镜，避免皮肤、黏膜接触以及吸入蒸气；戊二醛需避光保存，现配现用为佳。
5. 醛基活化后的磁珠稳定性有限，活化完成后应尽快加入蛋白进行偶联，不宜长时间放置。
6. 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)，建议将待偶联蛋白超滤换液至反应缓冲液中 。
7. 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。
8. 在正式实验前，需将试剂恢复至室温。
9. 操作过程中，务必穿戴实验服、一次性手套及防护装备。
10. 所有产品仅限于研究用途。
使用说明
1. 试剂准备
1.1 1 × 洗涤液配制：用超纯水将10 × 洗涤液稀释10倍，制备1 × 洗涤液。
1.2 活化缓冲液(含戊二醛)配制：活化缓冲液(含戊二醛)中戊二醛的终浓度为10%。对于25%的戊二醛，将1 × 洗涤液与25%的戊二醛按6:4的体积比例混合，例如0.6 mL 1 × 洗涤液加入0.4 mL 25%戊二醛，得到1 mL 活化缓冲液(含戊二醛)，可按实际使用量现用现配。25%或其它浓度的戊二醛需自备。
注意：活化缓冲液(含戊二醛)需现用现配，避光存放。
1.3 1 × PBST配制：用超纯水将10 × PBST稀释10倍，制备1 × PBST。
2. 氨基磁珠准备
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 × 洗涤液重悬磁珠，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. 氨基磁珠活化
3.1 向洗涤好的磁珠中加入100 μL新鲜配制的活化缓冲液(含戊二醛)，涡旋混匀使磁珠充分悬浮，25℃活化4-6 h。
注意：活化过程中磁珠需保持悬浮状态，活化完成后应尽快加入蛋白进行偶联，不宜长时间放置。
4. 氨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 用500 μL 1 × 洗涤液重悬磁珠，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。
4.3 偶联。加入100 μL(1-3.0 mg/mL)抗体溶液，用移液器轻轻吹打重悬磁珠，25℃偶联2 h，或者25℃偶联1 h后转入4℃过夜进行偶联。
注意：抗体需处于合适的缓冲液中，建议将待偶联蛋白超滤换液至反应缓冲液中，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500 μL 1 × 洗涤液重悬磁珠，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 用500 μL 1 × 洗涤液洗涤磁珠1次。
6.4 加入100 μL 保存液重悬磁珠，保存于2-8℃。
The UltraBio™ Amine Magnetic Bead Conjugation Kit manufactured by Aladdin supplies high-quality UltraBio™ Amine Magnetic Beads (UltraBio™ Amine Magnetic Beads). It adopts the classic glutaraldehyde crosslinking activation scheme, which can covalently immobilize primary amine-containing biological ligands such as antibodies, proteins and peptides onto the bead surface. 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, the operation is simple, and the conjugation reaction can be carried out at room temperature. Users only need to prepare glutaraldehyde and 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 amine 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 1 mg of IgG antibody. The maximum binding capacity depends on factors such as the molecular weight of the biomolecule. Glutaraldehyde crosslinking is performed under mild conditions and is friendly to protein activity. 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: Glutaraldehyde is a homobifunctional crosslinker. In aqueous solution, glutaraldehyde can self-polymerize to form polyaldehyde structures. One aldehyde group of polyglutaraldehyde reacts with amino groups on the surface of amine magnetic beads, while another aldehyde group further crosslinks with primary amino groups on ligands such as antibodies, proteins and peptides to form stable covalent linkages. After conjugation, blocking treatment quenches residual active aldehyde groups on the bead surface to reduce non-specific adsorption of the magnetic beads.
Contents and storage
| U1508966 | Component | Appearance | 50 reactions | Storage | Quantity Per reaction |
| U1508966A | Amine Magnetic Beads(1.2μm) | Brownish-black suspension | 5 mL | 2-8℃. Do not freeze. | 100 μL for 1 reaction |
| U1508966B | 10×Wash Buffer | liquid | 30 mL | 2-8℃. | Prepare according to instructions |
| U1508966C | Reaction Buffer | liquid | 10 mL | 2-8℃. | Use according to instructions |
| U1508966D | 10×PBST | liquid | 10 mL | 2-8℃. | Prepare according to instructions |
| U1508966E | Blocking Solution | liquid | 25 mL | 2-8℃. | Use according to instructions |
| U1508966F | 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. Glutaraldehyde solution.
3. Magnetic Rack.
4. 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. Amine 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. Glutaraldehyde is irritant and volatile. Perform all operations in a fume hood with gloves and goggles to avoid skin/mucosa contact and vapor inhalation. Store glutaraldehyde away from light and prepare freshly before use.
5. Aldehyde-activated beads have limited stability. Add target protein immediately after activation; prolonged storage of activated beads is not recommended.
6. Proteins intended for conjugation should not contain protein stabilizers including BSA. Buffers must be free of amine-containing compounds (e.g., Tris, glycine). Buffer exchange into Reaction Buffer by ultrafiltration is recommended.
7. 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.
8. Before formal experimentation, the reagents must be returned to room temperature.
9. During operation, always wear a lab coat, disposable gloves, and protective equipment.
10. All products are for research use only.
Instruction for use
1. Reagent Preparation
1.1 Prepare 1 × Wash Buffer: Dilute the 10 × Wash Buffer:10-fold with ultrapure water to prepare 1 × Wash Buffer:.
1.2 Prepare Activation Buffer(containing glutaraldehyde): The target final concentration of glutaraldehyde in this activation buffer is 10%. When using 25% glutaraldehyde solution, mix 1 × Wash Buffer and 25% glutaraldehyde solution in a 6:4 volume ratio. For example, combine 0.6 mL of 1 × Wash Buffer with 0.4 mL of 25% glutaraldehyde solution to yield 1 mL of activation buffer. Prepare only the volume required for immediate use. Glutaraldehyde solution (25% or other concentrations) is supplied by the user.
Note: Prepare the Activation Buffer(containing glutaraldehyde) immediately before use and protect it from light.
1.3 Preparation 1 × PBST: Dilute 10 × PBST 10-fold with ultrapure water to prepare 1 × PBST.
2. Amine 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 × Wash 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. Amine Magnetic Bead Activation
3.1 Add 100 μL of freshly prepared Activation Buffer(containing glutaraldehyde) to the washed magnetic beads. Vortex to fully suspend the beads, then incubate at 25℃ for 4-6 h for activation.
Note: The beads must remain suspended during activation. Add target protein immediately after activation; prolonged storage of activated beads is not recommended.
4. Conjugation of Biomolecules to Amine Magnetic Beads (demonstrated with antibody)
4.1 Magnetic separation. Place the beads on a magnetic rack for separation and remove the supernatant.
4.2 Resuspend the beads in 500 μL of 1 × Wash Buffer by gentle pipetting. Place the tube on a magnetic rack for magnetic separation and remove the supernatant. Repeat this procedure twice.
4.3 Conjugation. Add 100 μL of antibody solution (1-3.0 mg/mL). Gently resuspend the beads by pipetting, then incubate for conjugation at 25℃ for 2 h. Alternatively, incubate at 25 °C for 1 h followed by overnight conjugation at 4 °C.
Note: The antibody must be in a suitable buffer. Buffer exchange into Reaction Buffer by ultrafiltration is recommended. 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 500 μL of 1 × Wash 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 Wash the beads once with 500 μL of 1× Wash Buffer.
6.4 Resuspend the beads in 100 μL of storage buffer and store at 2-8℃.
| U1508966 | 组分 | 物理外观 | 50 reactions | 储存温度 | 每次反应用量 |
| U1508966A | 氨基磁珠(1.2μm) | 棕黑色混悬液 | 5 mL | 2-8℃，禁止冷冻 | 每次反应需使用100μL |
| U1508966B | 10×洗涤液 | 液体 | 30 mL | 2-8℃. | 请按说明配制 |
| U1508966C | 反应缓冲液 | 液体 | 10 mL | 2-8℃. | Use according to instructions |
| U1508966D | 10×PBST | 液体 | 10 mL | 2-8℃. | 请按说明配制 |
| U1508966E | 封闭液 | 液体 | 25 mL | 2-8℃. | Use according to instructions |
| U1508966F | 保存液 | 液体 | 5 mL | 2-8℃. | 每次反应需使用100μL |
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