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。
溴化氰活化亲和层析介质是一种CNBr预活化的琼脂糖介质，基质为4%高度交联琼脂糖，具备良好的物理和化学稳定性；介质常用于偶联抗体或含伯氨基的高分子量蛋白，偶联反应自发且迅速，偶联后的介质亲和性质稳定。 主要应用于蛋白、肽和核酸的偶联和固定 。
阿拉丁溴化氰活化亲和层析介质储存在丙酮中，凝胶和保护液的体积比为1:1，我司产品规格为实际凝胶的体积。
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使用方法
为充分保证介质的活性，本品使用100%丙酮 保存，请置于 2-8 ℃进行长期保存，短时运输、使用时可在常温进行。
1.溶液配置
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2、配基预处理：通常1 mL介质可偶联约5-10 mg蛋白配基。干粉状蛋白配基可用Solution B 溶解，溶液状态的蛋白配基则需将其缓冲液置换为Solution B。
3、介质预处理：取适量丙酮中保存的亲和层析介质放于玻璃砂芯漏斗中，用15 倍胶体积预冷Solution A 洗涤。
4、偶联配基洗涤完成后，将介质转移至配基溶液中（介质与配基溶液的体积比为 1:1），混匀 。 室温下摇床反应 2-4 h，或4℃反应过夜。
5、 清洗：反应结束后用5倍介质体积的Solution B进行清洗，去除未与介质偶联的蛋白配基。
6、 封闭：将清洗后的介质再用3倍介质体积的Solution C进行清洗并使用该溶液对介质剩余活化位点 进行封闭（介质与溶液的体积比为 1:1），室温摇床混合2-4 h。
7、交替清洗：封闭完成后用 Solution D与 Solution E交替洗涤 3-6次，每次用3倍介质体积清洗 。
8、 保存：最后再用10倍介质体积Solution F洗涤（可根据实际使用情况 换成其他结合缓冲液进行洗涤）。至此，配基偶联工作已经完成，该亲和介质即可使用。若要进行长期保存，请将介质置于 2-8 ℃、20%乙醇溶液中。
偶联影响因素
1、 偶联缓冲液：碳酸氢盐或硼酸盐缓冲液是常用的偶联溶液。勿使用 Tris或其他含氨基
的缓冲液作为偶联缓冲液；为保持较高的偶联效率，pH通常选择8-10；为减小蛋白之间的相互吸附，可在偶联溶液中加入适当浓度的盐，如0.5 M NaCl。
2、 温度与反应时间：室温条件下偶联反应 2-3 h内即可完成。若偶联需要在低温条件下（如 4 ℃）进行，可过夜反应（或反应 10-16 h）。
3、 配基浓度：建议每毫升介质加入 5-10 mg蛋白配基。配基添加量过多可能会导致介质
配基密度过高，而出现空间位阻效应，降低介质吸附量。同时，配基密度过高还会增加洗脱难度以及介质的非特异性吸附。
注：低pH会影响介质的偶联活性，但有时为减小空间位阻效应或保留配基生物学活性，交联反应需在低 pH下进行，如pH 6。
再生
常用再生方法如下：高pH缓冲液（如 0.1 M Tris 0.5 M NaCl pH 8.5 与 低 pH缓冲液（如 0.1 M NaAc 0.5 M NaCl pH=4.5 交替洗涤3-5次每次用 2-3倍 介质体积溶液洗涤。最后用结合缓冲液平衡。
注：再生方法因偶联配基而异，此方法仅供参考。
清洗
介质在使用3-5个循环后需要进行清洗，以维持介质的性能。对于不同类型的杂质或污染物，建议清洗剂选择如下：
去除微生物：常用的清洗剂有NaCl、过滤灭菌的缓冲液以及非离子型去污剂。
去除强结合蛋白：含非离子型去污剂的pH 8.5缓冲液或20%-70%乙醇。
储存条件
2-8℃，100% 丙酮
CNBr-activated Agrose is a pre-activated agarose medium. The matrix is 4% highly cross-linked agarose with good physical and chemical stability. This medium is commonly used for coupling antibodies or high-molecular-weight proteins containing primary amino groups. The coupling reaction is spontaneous and rapid, and the coupled medium exhibits stable affinity properties. It is mainly used for the conjugation and immobilization of proteins, peptides and nucleic acids.
Aladdin CNBr-activated Agrose is stored in acetone at a 1:1 volume ratio of gel to protective solution. The product specification refers to the volume of the actual gel.
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Instructions for Use
To ensure full activity, this product is stored in 100% acetone at 2–8 °C for long-term storage. Short-term transportation and use are allowed at room temperature.
1.Solution Preparation
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2.Ligand Pretreatment
Typically, 1 mL medium can couple approximately 5–10 mg protein ligand.
Powdered protein ligand: dissolve in Solution B.
Liquid protein ligand: exchange buffer to Solution B before use.
3.Medium Pretreatment
Place an appropriate amount of affinity chromatography medium (stored in acetone) in a glass sintered funnel. Wash with 15× gel volume of pre-chilled Solution A.
4.Ligand Coupling
After washing, transfer the medium into the ligand solution at a 1:1 volume ratio and mix well.ncubate on a shaker at room temperature for 2–4 h, or at 4 °C overnight.
5.Washing
After reaction, wash with 5× medium volume of Solution B to remove uncoupled protein ligand.
6.Blocking
Wash the medium with 3× medium volume of Solution C, then block remaining activated sites with Solution C at a 1:1 volume ratio.Incubate on a shaker at room temperature for 2–4 h.
7.Alternate Washing
After blocking, wash alternately with Solution D and Solution E for 3–6 cycles, using 3× medium volume per wash.
8.Storage
Finally, wash with 10× medium volume of Solution F (other binding buffers may be used as needed).Ligand coupling is complete and the affinity medium is ready for use.For long-term storage, store the medium in 20% ethanol at 2–8 °C.
Factors Affecting Coupling
1.Coupling BufferBicarbonate or borate buffers are commonly used. Do not use Tris or other amino-containing buffers as coupling buffers.To maintain high coupling efficiency, pH is usually 8–10.To reduce protein–protein adsorption, add an appropriate salt concentration (e.g.0.5 M NaCl).
2.Temperature and Reaction TimeCoupling is completed within 2–3 h at room temperature.For low-temperature coupling (e.g.4 °C), incubate overnight (10–16 h).
3.Ligand ConcentrationRecommended loading: 5–10 mg protein ligand per mL medium.Excess ligand may cause high ligand density, leading to steric hindrance, reduced binding capacity, difficult elution, and increased non-specific adsorption.
Note: Low pH reduces coupling activity. However, to minimize steric hindrance or preserve ligand bioactivity, coupling may be performed at lower pH (e.g.pH 6).
Regeneration
A typical regeneration procedure:Wash alternately with high-pH buffer (e.g.0.1 M Tris, 0.5 M NaCl, pH 8.5) and low-pH buffer (e.g. 0.1 M NaAc, 0.5 M NaCl, pH 4.5) for 3–5 cycles, using 2–3× medium volume per wash.Finally equilibrate with binding buffer.
Note: Regeneration varies by ligand; this method is for reference only.
Cleaning
The medium requires cleaning after 3–5 cycles to maintain performance.
Recommended cleaning agents for different contaminants:
Microorganisms: NaCl, filter-sterilized buffers, non-ionic detergents.
Tightly bound proteins: pH 8.5 buffer with non-ionic detergent or 20%–70% ethanol.
Storage Conditions
2–8 °C, 100% acetone
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636659 |
分析证书 | C1523223 |
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