计算溶液所需的质量、体积或浓度。
His标签蛋白纯化琼脂糖磁珠 (IDA-Co)
有货
为什么选择此级别
BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,禁止冷冻。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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。
文献证明
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。
概述
His标签蛋白纯化琼脂糖磁珠 (IDA-Co)区别于传统镍螯合磁珠，钴离子体系非特异性吸附更低，可获得更高纯度目的蛋白。产品依托磁性分离技术，无需层析柱、大型液相设备，粗蛋白样品无需长时间离心、过滤，仅搭配磁力架即可完成结合、洗涤、洗脱全流程操作，整套纯化流程 1 小时内完成，支持高通量多样品平行处理，大幅节省实验时间与耗材成本。
多孔琼脂糖骨架具备丰富活性结合位点，蛋白载量表现优异，磁珠悬浮性好、磁响应速度快，批次间性能稳定；适配细菌、酵母、昆虫细胞、哺乳动物细胞胞内 / 分泌表达的可溶性 His 标签蛋白，同时兼容包涵体变性蛋白纯化场景，广泛应用于基础分子生物学、酶制剂制备、蛋白互作、药物前体蛋白小试制备等科研与中试场景。产品温和螯合体系可减少目标蛋白氧化变性，操作后磁珠可通过咪唑再生重复使用，储存便捷，适配手动操作与自动化移液工作站，是高纯度 His 标签蛋白快速分离的优选介质。
本产品每毫升磁珠悬浊液含约10% v/v琼脂糖磁珠沉淀。
包装规格： 10% v/v悬液
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注：
①：磁珠蛋白结合量与目标蛋白特性有关，此处仅做参考值
②：1 mL 磁珠悬液中包含 100 μL 磁珠
适用范围
适用于纯化细菌、酵母、昆虫和哺乳动物细胞等分泌或胞内表达的可溶性组氨酸标签蛋白，也可用于变性蛋白的纯化（包涵体需变性后再进行纯化）。
操作流程
1. 缓冲液的准备
目标蛋白与组氨酸标签蛋白纯化磁珠的结合性能将直接影响目标蛋白的纯化效率，而各种缓冲液配制也将在一定程度上影响目标蛋白的回收率和纯度。因此，在较大规模蛋白纯化之前，用户应该自行设计实验，筛选出适合纯化目标蛋白的缓冲液体系，主要包括 Binding Buffer、Washing Buffer、ElutionBuffer。
增加咪唑浓度进行洗脱是组氨酸标签纯化磁珠最常用的洗脱方法，首次使用时，在不确定最佳的洗脱咪唑浓度情况下，推荐在缓冲液中分别加入 10 mM、20 mM、50 mM、100 mM、200 mM、300 mM、400 mM、500 mM 咪唑，浓度从低到高分别洗脱，磁吸后收集蛋白上清液，之后通过 SDS-PAGE 电泳等方法鉴定洗脱
结果。
以下提供的缓冲液体系适用于多数组氨酸标签蛋白的纯化，供用户参考。
Binding Buffer： 20 mM Phosphate Buffer，500 mM NaCl，5~50 mM Imidazole，pH7.4
Washing Buffer： 20 mM Phosphate Buffer，500 mM NaCl，50~100 mM Imidazole，pH7.4
Elution Buffer： 20 mM Phosphate Buffer，500 mM NaCl，500 mM Imidazole，pH7.4
2. 样品处理
①： 大肠杆菌、酵母等细胞内表达蛋白：表达细胞用适量的 Binding Buffer 稀释，加入蛋白酶抑制剂（如终浓度为 1 mM 的 PMSF），重悬细胞，冰浴超声裂解细胞，即为粗蛋白样品。如果样品过于粘稠，可根据需要在粗样品中加入适量核酸酶，冰浴 30 min，以降解核酸。另外，用户也可以根据实际需要对蛋白样品进行离心操作。
②： 胞外表达蛋白：取胞外表达上清，用等量 Binding Buffer 稀释，即为粗蛋白样品。
③：动物细胞胞内表达蛋白：取适量动物细胞，先用适量 PBS 洗涤 1 次，弃上清；接着用适量含 1%（V/V）Triton X-100 或 1%（V/V） NP-40 的 Binding Buffer 重悬，加入蛋白酶抑制剂（如终浓度为 1 mM 的 PMSF），冰浴10 min，即为粗蛋白样品。
3. 磁珠预处理
一般情况下，磁珠的使用量是由用户根据目标蛋白产量和磁珠载量信息计算获得。例如：采用大肠杆菌表达某目标蛋白，500 mL 发酵液收获 2 g 湿重的菌体，通过预实验估算其目标蛋白产量为 10～20 mg，用户需要取 5 mL磁珠悬液用于目标蛋白的纯化。以下即以此为例进行详细说明：
①： 将产品置于漩涡混匀器上充分混匀，用移液器取 5 mL 磁珠悬液于 15 mL 离心管中，进行磁性分离*，弃上清液，从磁性分离器上取下离心管。
②： 加入 5 mL Binding Buffer 到上述装有磁珠的离心管中，上下翻转离心管数次，使磁珠重新悬浮；进行磁性分离，移去上清液。重复洗涤 2 次。
注*：在磁性分离过程中，为了减少磁珠在使用过程中的损耗，待溶液变澄清后，盖紧离心管盖子，保持离心管仍在磁性分离器上，手持磁性分离器与离心管上下翻转数次，使澄清的溶液涮洗离心管盖上残留的磁珠，静置片刻，使溶液重新变澄清；以下同。）
4. 目标蛋白与磁珠结合
①：用 10 mL Binding Buffer 悬浮 2 g 湿重的菌体，进行破碎和裂解之后，即为目标粗蛋白样品，加入到装有预处理磁珠的离心管中，将离心管置于漩涡混匀器振荡 15 s。
②：将离心管置于旋转混合仪上，室温旋转混合 20~30 min（如果需要，可以在 2~8℃ 的低温环境下，旋转混 1 h，防止目标蛋白降解）。
③：将离心管置于磁性分离器上进行磁性分离，移出上清液至新的离心管中以备后续检测，从磁性分离器上取下离心管进行后续洗涤步骤。
5. 磁珠洗涤
①：加入 10 mL Washing Buffer 到装有磁珠的离心管中，轻轻翻转离心管数次，使磁珠重新悬浮，磁性分离，移出清洗液到新的离心管中，以备取样检测。重复此步骤 1 次
②：加入 10 mL Washing Buffer 到装有磁珠的离心管，使磁珠重新悬浮，将磁珠悬液转移到新的离心管（避免原离心管壁上非特异性吸附蛋白污染目标蛋白），磁性分离，移出上清液到清洗液收集管。
6. 目标蛋白洗脱
①：可根据需要改变洗脱体积调整目标蛋白浓度，加入 2~10 mL Elution Buffer，轻轻翻转离心管数次，使磁珠悬浮，磁性分离，收集洗脱液到新的离心管中，即为纯化的目标蛋白样品。
②：如果需要，可以重复上述步骤 1 次，收集样品到新的离心管中，以检测目标蛋白是否洗脱完全。
7. 磁珠后处理
①：在装有磁珠的离心管中加入 5 mL Elution Buffer，上下翻转离心管数次，使磁珠悬浮，磁性分离，去除上清液。
②：重复上述步骤 2 次。
③：在离心管中加入 5 mL ddH₂O，上下翻转离心管数次，使磁珠悬浮，磁性分离，去除上清液。
④：重复上述步骤 2 次。
⑤：加入 Storage Buffer 到磁珠中使总体积为 5 mL，保存于 2~30℃（长期保存，置于 2~8℃），可用于下一次同种蛋白的纯化。
8. 磁珠再生
磁珠连续使用三次以上，其结合目标蛋白的能力可能会明显降低，建议进行磁珠再生处理。
Stripping Buffer：20 mM Sodium Phosphate，500 mM NaCl，100 mM EDTA， pH 7.4
Beads Washing Buffer（可选）：0.5 M NaOH，2 M NaCl
Recharge Buffer：100 mM NiSO₄ / CoCl₂（该化学试剂有一定的毒性，可能造成过敏反应，使用时务必注意）
Storage Buffer：20%（V/V）乙醇
以 5 mL 10%（V/V）磁珠悬液为例，详细说明磁珠再生操作：
①： 将磁珠悬液进行磁性分离，去除上清液，从磁性分离器上取下离心管，在离心管中加入 5 mLddH₂O， 上下翻转离心管数次，使磁珠重新悬浮，磁性分离，去除上清液。
② ：加入 5 mL Stripping Buffer，上下翻转离心管数次，使磁珠重新悬浮，室温旋转混合 5 min，磁性分离，去除上清液。重复此步骤 1 次。
③ ：加入 5 mL ddH₂O，上下翻转离心管数次，使磁珠重新悬浮，磁性分离，去除上清液，重复此步骤 2 次。
④ ：碱处理：加入 5 mL Beads Washing Buffer，上下翻转离心管数次，使磁珠重新悬浮，室温旋转混合 5 min，磁性分离，去除上清液。加入 5 mL ddH₂O，上下翻转离心管数次，使磁珠重新悬浮，磁性分离，去除上清液。重复 ddH₂O 洗涤步骤 3~5 次，至洗涤液呈中性为止。
⑤ ：加入 5 mL Recharge Buffer，上下翻转离心管数次，使磁珠重新悬浮，室温旋转混合 20 min，磁性分离，去除上清液。
⑥：加入 5 mL ddH₂O，上下翻转离心管数次，使磁珠重新悬浮，磁性分离，去除上清液。重复此步骤 4 次以上，保证镍离子去除完全。
⑦：加入 Storage Buffer 到磁珠中使总体积为 5 mL，保存于 2~30℃ （长期保存，置于 2~8℃）。
蛋白纯化流程的优化
以上操作流程适用于大部分组氨酸标签蛋白的纯化，根据目标蛋白与金属离子螯合磁珠的结合性能不同，用户可以从以下几个方面对纯化流程进行优化，以提高目标蛋白的回收率和纯度。提高目标蛋白回收率的参考方法：
1. 降低样品溶液和 Binding Buffer 中的 Imidazole 浓度；
2. 样品溶液和其它缓冲液中添加表面活性剂等物质；
3. 添加合适的蛋白酶抑制剂，防止目标蛋白降解；
4. 增加磁珠用量；
5. 延长蛋白与磁珠孵育的时间；
6. 延长洗脱目标蛋白的时间或增加洗脱次数。
2. 样品溶液和其它缓冲液中添加表面活性剂等物质；
3. 添加合适的蛋白酶抑制剂，防止目标蛋白降解；
4. 增加磁珠用量；
5. 延长蛋白与磁珠孵育的时间；
6. 延长洗脱目标蛋白的时间或增加洗脱次数。
提高目标蛋白纯度的参考方法：
1. 提高样品溶液和 Binding Buffer 中 Imidazole、NaCl 的浓度；
2. 样品和缓冲液中添加表面活性剂等物质；
3. 添加合适的蛋白酶抑制剂，防止目标蛋白降解；
4. 延长洗涤的时间，增加洗涤次数；
5. 采用梯度 Imidazole 浓度洗脱目标蛋白。
注意事项
1. 首次使用本产品前，请务必详细阅读本用户手册；
2. 磁珠使用和保存过程中应避免冷冻、干燥和高速离心等操作；
3. 在使用本产品前，请务必充分振荡使磁珠保持均匀的悬浮状态；
4. 请选用质量好的移液器吸头和离心管，以免磁珠贴壁或混合过程发生渗漏引起磁珠的损耗；
5. 磁珠与溶液混合过程中，如果溶液粘稠无法通过翻转离心管重悬磁珠，可采用移液器反复吹吸或短时漩涡混合使磁珠充分重悬；
6. 用户可根据实际需要保留经磁性分离移去的上清液，进行取样检测，以便分析纯化过程和优化蛋白纯化流程；
7. 本产品可以重复使用，当纯化性能降低时，建议进行再生处理；
8. 使用过的磁珠重复使用时，建议纯化同种蛋白，纯化不同种类的蛋白时，建议使用新的磁珠；
9. 本产品需与磁性分离器配套使用；
10. 本产品仅供研究使用。
His-tag Protein Purification Agarose Magnetic Beads (IDA-Co) differ from traditional nickel-chelated magnetic beads by utilizing a cobalt ion system that exhibits lower non-specific adsorption, thereby yielding target proteins of higher purity. Leveraging magnetic separation technology, this product eliminates the need for chromatography columns or large-scale liquid chromatography equipment. Crude protein samples do not require prolonged centrifugation or filtration. The entire purification process—including binding, washing, and elution—can be completed within 1 hour using just a magnetic stand. It supports high-throughput, parallel processing of multiple samples, significantly saving experimental time and consumable costs.
The porous agarose matrix provides abundant active binding sites, resulting in excellent protein loading capacity. The beads exhibit good suspendability, fast magnetic responsiveness, and consistent batch-to-batch performance. They are suitable for purifying soluble His-tagged proteins expressed intracellularly or secreted from bacteria, yeast, insect cells, and mammalian cells, and are also compatible with purification of denatured proteins from inclusion bodies. The product is widely used in basic molecular biology, enzyme preparation, protein-protein interaction studies, and small-scale preparative production of drug precursor proteins in both research and pilot-scale settings. The mild chelation system minimizes target protein oxidation and denaturation. After use, the beads can be regenerated with imidazole and reused, are easy to store, and are compatible with both manual operation and automated liquid handling workstations, making them a preferred medium for rapid isolation of high-purity His-tagged proteins.
Each milliliter of the magnetic bead suspension contains approximately 10% (v/v) agarose magnetic bead sediment.
Package Specification: 10% (v/v) suspension
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Notes:
①: The protein binding capacity is related to the characteristics of the target protein; this value is for reference only.
②: 1 mL of bead suspension contains 100 μL of beads.
Scope of Application
Suitable for purifying soluble histidine-tagged proteins secreted or intracellularly expressed in bacteria, yeast, insect cells, and mammalian cells. Also suitable for purifying denatured proteins (inclusion bodies must be denatured prior to purification).
Protocol
1. Buffer Preparation
The binding performance between the target protein and the His-tag purification magnetic beads directly affects purification efficiency. Various buffer compositions can also influence the recovery yield and purity of the target protein. Therefore, before large-scale protein purification, users should design their own experiments to screen for the optimal buffer system for their specific target protein, primarily including Binding Buffer, Washing Buffer, and Elution Buffer.
Increasing imidazole concentration for elution is the most common method. For first-time use without knowing the optimal imidazole concentration for elution, it is recommended to prepare elution buffers containing 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole. Elute sequentially from low to high concentration, collect protein supernatants after magnetic separation, and analyze elution results via SDS-PAGE or other methods.
The buffer systems provided below are suitable for purifying most histidine-tagged proteins and are for user reference.
Binding Buffer: 20 mM Phosphate Buffer, 500 mM NaCl, 5~50 mM Imidazole, pH 7.4
Washing Buffer: 20 mM Phosphate Buffer, 500 mM NaCl, 50~100 mM Imidazole, pH 7.4
Elution Buffer: 20 mM Phosphate Buffer, 500 mM NaCl, 500 mM Imidazole, pH 7.4
2. Sample Preparation
① Intracellularly expressed proteins in E. coli, yeast, etc.: Resuspend cell pellets in an appropriate volume of Binding Buffer. Add protease inhibitors (e.g., PMSF at a final concentration of 1 mM). Lyse cells by sonication on ice. This constitutes the crude protein sample. If the sample is too viscous, add nuclease as needed and incubate on ice for 30 min to degrade nucleic acids. Optionally, centrifuge the crude sample as needed.
② Extracellularly expressed proteins: Dilute the extracellular expression supernatant with an equal volume of Binding Buffer. This is the crude protein sample.
③ Intracellularly expressed proteins in animal cells: Wash an appropriate amount of cells once with PBS and discard the supernatant. Resuspend cells in Binding Buffer containing 1% (V/V) Triton X-100 or 1% (V/V) NP-40. Add protease inhibitors (e.g., PMSF at a final concentration of 1 mM). Incubate on ice for 10 min. This is the crude protein sample.
3. Bead Pre-treatment
Generally, the amount of beads used is calculated based on the target protein yield and bead binding capacity. Example: Using E. coli expressing a target protein, 2 g wet cell pellet from 500 mL culture yields approximately 10-20 mg target protein (estimated from preliminary experiments). The user needs to take 5 mL of bead suspension for purification. The procedure is detailed below using this example:
① Thoroughly resuspend the product by vortexing. Pipette 5 mL of bead suspension into a 15 mL centrifuge tube. Perform magnetic separation*, discard the supernatant, and remove the tube from the magnetic separator.
② Add 5 mL of Binding Buffer to the tube containing the beads. Invert the tube several times to resuspend the beads. Perform magnetic separation and discard the supernatant. Repeat this wash step twice.
(During magnetic separation, to minimize bead loss, after the solution clears, cap the tube, keep it on the magnetic separator, and gently invert the separator and tube together several times to rinse any beads stuck on the cap back into the solution. Let it stand briefly until the solution clears again. Same applies below.)
4. Target Protein Binding
① Resuspend 2 g wet cell pellet in 10 mL Binding Buffer. After cell disruption and lysis (the crude target protein sample), add this sample to the tube containing the pre-treated beads. Vortex the tube for 15 seconds.
② Incubate the tube on a rotator mixer for 20-30 minutes at room temperature. (If necessary, to prevent target protein degradation, incubate for 1 hour at 2-8°C).
③ Place the tube on the magnetic separator for separation. Transfer the supernatant to a new tube for later analysis if needed. Remove the tube with the beads from the separator for the subsequent washing steps.
5. Bead Washing
① Add 10 mL of Washing Buffer to the tube containing the beads. Gently invert the tube several times to resuspend the beads. Perform magnetic separation and transfer the wash solution to a new tube for sampling/analysis if needed. Repeat this step once.
② Add 10 mL of Washing Buffer to the beads, resuspend them, and transfer the entire bead suspension to a new centrifuge tube (to avoid contamination of the target protein with proteins non-specifically adsorbed to the original tube wall). Perform magnetic separation and transfer the supernatant to the wash solution collection tube.
6. Target Protein Elution
① Adjust elution volume as needed to concentrate the target protein. Add 2-10 mL of Elution Buffer. Gently invert the tube several times to resuspend the beads. Perform magnetic separation and collect the eluate (purified target protein sample) in a new tube.
② If necessary, repeat step 1 once and collect the sample in a new tube to check if elution is complete.
7. Bead Post-Treatment
① Add 5 mL of Elution Buffer to the tube containing the beads. Invert the tube several times to resuspend the beads. Perform magnetic separation and discard the supernatant.
② Repeat the above step twice.
③ Add 5 mL of ddH₂O to the tube. Invert several times to resuspend the beads. Perform magnetic separation and discard the supernatant.
④ Repeat the above step twice.
⑤ Add Storage Buffer to the beads to a total volume of 5 mL. Store at 2-30°C (for long-term storage, store at 2-8°C). The beads are ready for purification of the same protein in the next experiment.
8. Bead Regeneration
After more than three consecutive uses, the binding capacity of the beads may decrease significantly; regeneration is recommended.
Stripping Buffer: 20 mM Sodium Phosphate, 500 mM NaCl, 100 mM EDTA, pH 7.4
Beads Washing Buffer (Optional): 0.5 M NaOH, 2 M NaCl
Recharge Buffer: 100 mM NiSO₄ / CoCl₂ (Note: These chemicals are toxic and may cause allergic reactions; handle with care)
Storage Buffer: 20% (V/V) Ethanol
Example for regenerating 5 mL of 10% (V/V) bead suspension:
① Perform magnetic separation on the bead suspension, discard the supernatant. Remove tube from separator. Add 5 mL ddH₂O, invert several times to resuspend beads. Magnetize, discard supernatant.
② Add 5 mL Stripping Buffer, invert to resuspend, rotate mix at room temperature for 5 min, magnetize, discard supernatant. Repeat once.
③ Add 5 mL ddH₂O, invert to resuspend, magnetize, discard supernatant. Repeat twice.
④ Alkali treatment: Add 5 mL Beads Washing Buffer, invert to resuspend, rotate mix at room temperature for 5 min, magnetize, discard supernatant. Add 5 mL ddH₂O, invert to resuspend, magnetize, discard supernatant. Repeat ddH₂O wash steps 3-5 times until the wash solution is neutral.
⑤ Add 5 mL Recharge Buffer, invert to resuspend, rotate mix at room temperature for 20 min, magnetize, discard supernatant.
⑥ Add 5 mL ddH₂O, invert to resuspend, magnetize, discard supernatant. Repeat this step at least 4 times to ensure complete removal of excess nickel/cobalt ions.
⑦ Add Storage Buffer to the beads to a total volume of 5 mL. Store at 2-30°C (for long-term storage, store at 2-8°C).
Optimization of Protein Purification Protocol
The protocol above is suitable for purifying most histidine-tagged proteins. Depending on the binding characteristics of the target protein to the metal-chelated beads, users can optimize the protocol from the following aspects to improve recovery and purity.
Methods to Improve Target Protein Recovery:
1. Reduce imidazole concentration in the sample solution and Binding Buffer.
2. Add surfactants or other agents to the sample and buffers.
3. Add appropriate protease inhibitors to prevent target protein degradation.
4. Increase the amount of beads used.
5. Extend the incubation time between protein and beads.
6. Extend the elution time or increase the number of elution steps.
Methods to Improve Target Protein Purity:
1. Increase imidazole and NaCl concentrations in the sample solution and Binding Buffer.
2. Add surfactants or other agents to the sample and buffers.
3. Add appropriate protease inhibitors to prevent target protein degradation.
4. Extend washing time and increase the number of wash steps.
5. Use a gradient imidazole concentration for elution.
Precautions
1 Before using this product for the first time, be sure to read this user manual thoroughly.
2 During use and storage, avoid freezing, drying, or high‑speed centrifugation of the beads.
3 Before using this product, always shake vigorously to ensure the beads are in a uniform suspension.
4 Use high‑quality pipette tips and centrifuge tubes to prevent bead loss due to adhesion to tube walls or leakage during mixing.
5 If the solution is viscous and inversion of the tube does not resuspend the beads during mixing, use repeated pipetting or brief vortex mixing to ensure complete resuspension.
6 Users may keep the supernatant removed by magnetic separation for sampling and analysis, as needed, to evaluate the purification process and optimize the protein purification protocol.
7 This product can be reused. When purification performance decreases, regeneration is recommended.
8 When reusing used beads, it is recommended to purify the same protein. For purifying different proteins, use fresh beads.
9 This product must be used with a magnetic separator.
10 This product is for research use only.
规格
产品名称
His标签蛋白纯化琼脂糖磁珠 (IDA-Co)
规格或纯度
BioReagent, 10% v/v
产品线
生物产品
储存与运输
物理形态
液体
储存缓冲液
20% Ethanol
浓度
10% v/v
储存条件
2-8°C储存,禁止冷冻
运输条件
低温运输
稳定性与储存
Store at 2-8℃ long term (24 months). Do not freeze.
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
溶液计算器
常见问题
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品应如何储存？
请于2–8 °C条件下保存。请勿冷冻。冷冻可能对本品造成不可逆的损坏。
本产品如何运输？
本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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危险品化学品经营许可证（带存储）