BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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。
钥孔戚血蓝蛋白(mcKLH)是高质量形式的KLH，从海水养殖生长的帽贝中获得，其免疫原性与KLH相似，但作为于生产抗半抗原抗体的载体蛋白，具有更高的稳定性和效率。每个mcKLH分子可偶联多个抗原分子，以此提升抗原免疫原性，提高免疫动物针对目标表位产生高滴度抗血清的概率，同时mcKLH具有高分子质量（ 4.5×10⁵至 1.3×10⁷ 道尔顿；350和 390 kDa 亚基聚集体），可引发比BSA或OVA更强的免疫应答。因此常作为载体蛋白，与半抗原、弱免疫原偶联，用于动物免疫制备特异性抗体。
本试剂盒采用EDC（1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐）杂双功能交联剂实现半抗原与载体蛋白偶联，所得偶联物可刺激机体产生免疫应答，制备靶向半抗原的抗体。EDC能够与半抗原表面暴露的羧基、氨基发生反应，形成稳定化学键。试剂盒包含mcKLH以及适配碳二亚胺反应的专用缓冲液，同时配备脱盐离心柱，仅需简单离心即可实现蛋白的高效回收，每套可支持1次或5次独立偶联反应。
产品组份
|
其他所需试剂
1. 离心机。
2. 用于标记的半抗原。
3. PBS缓冲液（pH 7.2-7.4）。
重要须知
1. 本方案为通用流程，适用于绝大多数半抗原；需根据半抗原分子量、空间结构优化条件以提升偶联效率。
2. 体系中半抗原摩尔量过量可保证偶联充分；通常半抗原与mcKLH等质量投料即可实现摩尔过量。
3. 反应体系出现沉淀属于正常现象，原因为半抗原与载体蛋白偶联过度；减少EDC用量可减轻沉淀。
4. 反应体系添加过DMSO时，平衡柱子的纯化缓冲液需加入同等比例DMSO，避免脱盐过程蛋白析出。
5. 若偶联反应出现沉淀：离心收集上清单独纯化，沉淀留存；纯化完成后将沉淀与纯化液合并使用。
6. 纯化填料建议一次性使用。
7. 在正式实验前，需将试剂恢复至室温，在使用前将试剂进行离心，较低的温度和离心管壁试剂残留均会影响偶联效果；
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. 配制mcKLH溶液（10 mg/mL）：向mcKLH中加入200 μL偶联缓冲液，配制成10 mg/mL mcKLH溶液。
2. 制备半抗原溶液（5 mg/mL）：取2 mg半抗原，溶于400 μL偶联缓冲液。
注意：部分半抗原水溶性差，可用DMSO助溶，DMSO在终反应体系中占比＜30%；过高浓度DMSO会造成mcKLH不可逆变性。
3. 将400 μL半抗原溶液全部加入到200 μL mcKLH溶液中，混匀得到半抗原-mcKLH混合液。
4. 向一管EDC中加1 mL纯水，立即取50 µL EDC溶液加入到半抗原-mcKLH混合液中，轻轻混匀。室温孵育2小时。
注意：反应进行期间，可提前进行步骤5的操作，完成脱盐柱的平衡。
5. 脱盐柱预平衡
5.1 将一个空的离心柱放入一个 15 mL 收集管中。
5.2 通过反复倒置试剂瓶重悬纯化树脂填料。然后取3.0 mL悬液加入柱中，让树脂沉降。让柱缓冲液依靠重力从柱中排出。初始时，可能需要施加一些压力使最初的几滴缓冲液流出。以1000 × g离心柱子2分钟，弃去储存缓冲液，将柱子放回同一个收集管中。
注意：纯化树脂以20%乙醇的悬液形式提供，树脂与乙醇的体积比为3:1(v/v)。
5.3 向脱盐柱树脂顶部加入2 mL PBS，1000×g离心2分钟，弃去流出液；
注意：若偶联物短期1周内使用：可使用PBS完成脱盐；如需冻存，必须使用试剂盒配套的纯化缓冲液，可避免冻融过程蛋白失活析出
5.4 重复步骤5.3两次，第三次洗涤时加入PBS，直到进入下一步骤时再进行离心；
注意：使用定角转子时，在柱子背对转子中心的一侧做标记。在后续所有离心步骤中，将柱子放入离心机时，标记面应背对转子中心。
5.5 第三次洗涤离心后，脱盐柱中应无溶液残留，如有残留，可短暂离心直至溶液完全排出；
6. 偶联物脱盐纯化
6.1 将准备好的脱盐柱放入新的15 mL离心管中，并将步骤4中的全部样品加入脱盐柱中；
注意：若反应出现沉淀，离心收集上清单独纯化，沉淀留存；纯化完成后将沉淀与纯化液合并使用。
6.2 脱盐柱1000 × g离心2分钟，收集管中的即为纯化偶联物。
7. 保存
7.1 纯化后可直接用于动物免疫；若存放超过数日，无菌过滤后置于无菌容器，4℃短期保存或分装-20℃冻存。
mcKLH is a high-quality form of KLH that is harvested from limpets grown in mariculture. It exhibits immunogenicity comparable to KLH, yet provides enhanced stability and efficiency as a carrier protein for generating anti-hapten antibodies. Several antigen molecules can be conjugated to each mcKLH molecule to increase their immunogenicity and likelihood of eliciting immunized animals to produce high-titer antisera to the epitopes of interest. Meanwhile, KLH has a high molecular mass (0.5 to 13 million daltons; aggregates of 350 and 390 kDa subunits) and elicits a stronger immune response than BSA or OVA. Consequently, mcKLH is a popular carrier protein for conjugation to haptens and other weak antigens to make them more immunogenic for antibody production.
This kit is designed for the coupling of haptens to carrier proteins, utilizing the primary amine groups of the hapten. This kit utilizes the chemical heterobifunctional crosslinker EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). The resulting conjugates can stimulate immune responses in organisms for the production of hapten-targeted antibodies. The carboxyl-reactive carbodiimide reacts with exposed carboxyl and amino groups on haptens to form stable bonds. This kit contains mcKLH and dedicated buffers compatible with carbodiimide reactions, as well as desalt spin columns that enable highly efficient protein recovery via a simple centrifugation step. This kit is suitable for 1 or 5 coupling reactions.
Contents and storage
|
Required materials not supplied
1. Microcentrifuge capable of 1000 × g.
2. Desired hapten for labeling.
3. PBS buffer (pH 7.2-7.4).
Important
1. The following protocol is a general protocol for a wide variety of haptens. Optimize the protocol accordingly for the conjugation efficiencies based on the size and structure of your hapten.
2. Using a molar excess of hapten over carrier protein ensures efficient conjugation. In general, a reaction with equal mass amounts of hapten and carrier protein will achieve sufficient molar excess.
3. In some cases precipitation may occur. The precipitation occurs due to excessive hapten-carrier protein conjugation. The precipitation may be minimized by reducing the amount of EDC in the reaction.
4. If DMSO was used in the conjugation, prepare the Purification Buffer Salts with the same percentage of DMSO used for the conjugation. This minimizes precipitation in the column during desalting.
5. If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.
6. Do not reuse the purification resin.
7. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.
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. Prepare mcKLH solution (10 mg/mL): Add 200 µL of EDC Conjugation Buffer into the vial of mcKLH to make a 10 mg/mL solution.
2. Prepare hapten solution (5 mg/mL): Dissolve 2 mg hapten in 400 µL EDC Conjugation Buffer.
Note: For haptens with limited solubility, use ＜ 30% DMSO in the final conjugation solution. Using a higher concentration of DMSO might irreversibly denature the carrier protein.
3. Add the 400 μL of hapten solution to the 200 μL carrier protein solution to have Hapten-mcKLH mixture solution.
4. Add 1 mL of ultrapure water to one vial of EDC. Immediately transfer 50 µL of the EDC solution into the hapten-mcKLH mixture and mix gently. Incubate at room temperature for 2 hours.
Note: During the incubation period, proceed to steps 5 below, to prepare a spin column for the purification of the labeled protein.
5. Prepare the spin column
5.1 Place an empty spin column in a 15 mL collection tube.
5.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 3.0 mL of the suspension into the column and allow the resin to settle. Allow the column buffer to drain from the column by gravity. Initially, some pressure may be required to cause the first few drops of buffer to elute. Centrifuge the column at 1000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube.
Note: The purification resin is supplied as a slurry in 20% ethanol, with a resin-to-ethanol volume ratio of 3:1 (v/v).
5.3 Add 2 mL of PBS Buffer, then centrifuge the column-tube assembly at 1000 × g for 2 minutes to equilibrate the column.
Note: If the conjugate is to be used for injection within one week, PBS may be used for purification. If the conjugate will be frozen, use the Imject Purification Buffer Salts for purification, which will preserve the product during freeze-thaw cycles.
5.4 Repeat step 5.3 for two additional times. Add PBS for the third wash but wait to centrifuge until immediately prior to proceeding to step.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
5.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
6. Purification with Desalting Column
6.1 Place column into a new 15 mL collection tube and apply the entire sample from step 4 to the column.
Note: If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.
6.2 Centrifuge the column-tube assembly at 1000 × g for 2 minutes. The purified conjugate is in the collection tube.
7. Strorage
7.1 The hapten-carrier conjugate can now be used for immunization. If the hapten-carrier conjugate is to be stored for more than a few days, sterile filter the conjugate and store in a sterile container at 4℃ or -20℃
| M1509082 | 组分 | 物理外观 | 1 reaction | 5 reactions | 储存温度 | 每次反应用量 |
| M1509082A | 血蓝蛋白(mcKLH) | 冻干粉 | 2 mg | 5×2 mg | 2-8℃，禁止冷冻 | 2mg mcKLH可用于偶联2mg的半抗原 |
| M1509082B | EDC | 白色粉末 | 10 mg | 5×10 mg | 2-8℃. | 请按说明配制 |
| M1509082C | EDC偶联缓冲液 | 无色透明液体 | 4 mL | 20 mL | 2-8℃. | 请按说明配制 |
| M1509082D | 纯化缓冲液 | 无色透明液体 | 10 mL | 50 mL | 2-8℃. | 请按说明配制 |
| M1509082E | 纯化填料 | 白色微珠混悬液 | 3 mL | 15 mL | 2-8℃，禁止冷冻 | 每次反应需使用3mL |
| M1509082F | 离心柱管 | 透明管体 | 1 EA | 5 EA | 2-8℃. | 每次反应需使用1套 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939000 |
分析证书 | M1509082 |
| ZJ26F0939001 |
分析证书 | M1509082 |
危险品化学品经营许可证（带存储）