本产品是羧甲基天冬氨酸（CM-Asp）基团螯合钴离子的金属螯合填料，主要用于带组氨酸标签（His-tag）蛋白的亲和层析。与同是金属螯合四配位的次氮基三乙酸（NTA）配基相比，CM-Asp 螯合钴离子后的最大特点是高选择性。结合配基修饰过程的优化，该产品做到了亲和力和特异性的平衡：对大部分带组氨酸标签蛋白的结合量可达20 mg/mL 的同时，例如对带氨酸标签的重组蛋白G、链霉亲和素的动态结合载量DBC10%分别可达25、35 mg/mL，一步纯化后样品纯度可达90%以上。
综合配基和基球的特点，Co羧甲基天冬氨酸亲和层析介质 (His标签) 适用于对纯度要求高或者Ni2+螯合填料无法满足纯度要求样品的工艺筛选和放大生产过程。
阿拉丁Co羧甲基天冬氨酸亲和层析介质储存在20%乙醇中，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。
参数 | 指标 | 基质 | 高刚性琼脂糖 | 配基 | 羧甲基天冬氨酸，预螯合~17 μmol Co2+/ml | 粒径范围 ① | 30~100 μm | 平均粒径 | ~60 μm | 动态结合载量 ② | ~20 mg/mL 带组氨酸标签蛋白 | 推荐工作流速 | 60~300 cm/h | 最大流速与压力 ③ | 1500 cm/h，0.5 MPa | 使用pH | 3~12（推荐的工作pH），3~12（长期稳定）；2~14（短期稳定）④ | 化学稳定性 | 在以下溶液中稳定：常用的水相缓冲液、8 M 尿素、6 M 盐酸胍、70%乙醇、10 mM β-巯基乙醇等。不推荐长时间在β-巯基乙醇等还原剂条件下使用。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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注：
① 90%体积以上的微球在此粒径范围；
② DBC10%测试条件为：10%流穿，大肠杆菌表达带组氨酸标签的重组蛋白，6 min停留时间；
③ 10 cm 柱高下的最大测试流速;
④ 剥离金属离子后CIP过程的pH 稳定性。
使用说明
1、色谱柱装填
以下阐述与层析系统连接时，填料的色谱柱装填方法。
（1）所有需要用到的材料的温度要与色谱操作的温度一样，液体最好做脱气处理。
（2）填料用量计算：通过沉降定量填料体积，需要的沉降填料体积=柱体积×压缩比（也称压缩因子）。沉降体积是指填料在20%乙醇保存液中自然沉降完全读取的稳定体积。Co羧甲基天冬氨酸亲和层析介质的压缩比为~1.10。为使达到压缩比，可通过柱头下压的方法，也可以通过高流速压柱。
（3）填料清洗：将填料悬液充分摇匀后量取相应体积，抽去液体，并用约3倍填料体积的纯化水洗涤，重复3次，以除去保存液。
（4）装柱填料悬液准备：将填料转移到适当的容器中，加入适量的装柱液，制成50~75 v%的装柱填料悬液（原包装中填料体积分数为50%），使用前搅匀。
（5）装填前准备：在清洗干净的层析柱下端加入装柱液，以除去下垫片及层析柱下端的空气，在柱内保留少量的蒸馏水，拧紧下堵头，调整柱子使其垂直于地面。
（6）装填：将搅匀后的填料悬液一次性缓慢倒入层析柱内（必要时使用装柱器），为避免引入气泡，应使之沿层析柱内壁自然流下。将所有填料加入后，用装柱液将装柱器加满，拧紧装柱器上盖，将柱子与层析系统连接。
（7）压柱：使柱内填料自然沉降（或50~150 cm/h 的低流速下沉降），待填料沉降完全后（填料与液体的界面清晰），去除装柱器，装上上柱头，并将柱头下降至界面处；通过高流速（推荐流速见下表，注意柱压不超过0.3 MPa），继续压柱至界面清晰稳定，标记界面稳定时的柱高。停泵，打开柱头上的阀门/堵头，关闭柱底的阀门/堵头，下压柱头至标记位置下方与压缩比对应的位置，旋紧柱头，装柱完成。装柱完成后，需用高流速平衡柱内的填料。
装柱条件 | Co羧甲基天冬氨酸亲和层析介质 | 压缩比 | ~1.10 | 装柱流速 | ~600 cm/h |
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2、柱效测定和评价
完成装柱后、使用前可通过柱效测定和评价可以确认层析柱装填质量。柱效通常用理论塔板高度（HETP）和非对称因子（As）来评价。柱效测定可以采用丙酮或者NaCl作为样品进行，按照下表配制样品溶液和流动相。
样品 | 1.0%丙酮 | 0.8~1.0 M NaCl | 样品体积 | 1.0%柱体积 | 1.0%柱体积 | 流动相 | 纯水 | 0.4 M NaCl | 流速 | 30 cm/h | 30 cm/h | 检测器 | UV-280 nm | 电导 |
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根据UV或者电导率曲线计算理论塔板高度（HETP）、理论塔板数（N）和非对称因子（As），公式如下：
HETP = L / N
N = 5.54 × (VR / Wh )2
As = a / b
其中：L为柱高； VR为保留体积；Wh为半高峰宽；a为在10%峰高处的第一个半峰宽；b为在10%峰高处的第二个半峰宽。
一般来说，HETP的数值应小于填料平均粒径的三倍（即HETP/D50<3，D50为填料的平均粒径），As应在0.8~1.5之间。
3、平衡与上样
金属螯合填料与磷酸盐、Tris-HCl 等缓冲体系兼容，例如Tris-HCl 或PB+150~500 mM NaCl（pH=7.4）的体系较为常用。初始的缓冲液称为平衡缓冲液，记为缓冲液A。Co羧甲基天冬氨酸亲和层析介质的缓冲液A通常不含咪唑，pH 在7~8 之间居多，呈弱碱性。
在上样前，需用缓冲液A 在操作流速下平衡层析柱，该过程通常需要5~10个柱体积的缓冲液A，以电导、pH 等检测信号参数不变且与缓冲液A对应为准。
上样量可按“mg 目标蛋白/mL 填料”来设定，通常为DBC10%的50~80%。除了DBC10%，也可以测试上样流穿液中的目标蛋白确定上样量。
上样后需要3~10个柱体积的缓冲液A再平衡层析柱。
4、洗杂
在缓冲液A 中添加5~40 mM 的咪唑冲洗层析柱，可以将非特异性结合的杂蛋白除去。咪唑浓度越高，杂蛋白被除去得越彻底，但会降低目标蛋白的收率。洗杂过程咪唑的浓度与不同蛋白的特性相关，可通过阶跃洗杂过程（例如5 mM、10 mM、20 mM……）快速优化洗杂条件。洗杂的体积通常为3~10个柱体积。
5、洗脱
最常用的洗脱方式为提高洗脱液中咪唑的浓度（在缓冲液A中加入咪唑），推荐在0~500 mM 范围内进行阶跃洗脱或者线性梯度洗脱。对于大多数带组氨酸标签的蛋白，200 mM的咪唑浓度可以将目标蛋白洗脱完全。对于一些场合，也可以改变pH，需注意pH不能低于4。
6、再生
在多次使用后，通常是5~7 次以上，需要对填料进行再生。可以选择0.5~1 柱体积200 mM EDTA+500 mM NaCl （pH=7）的缓冲液将金属离子剥离。再用50~200 mM的氯化钴溶液与CM-ASP 配基螯合，用纯水将过量的金属离子除去。
金属离子的重新螯合参照以下步骤。
(1) 配制50~200 mM 的氯化钴溶液，需注意控制不同pH条件下不同金属离子的溶解性，pH 过高可导致沉淀。
(2) 用纯水清洗色谱柱，纯水用量3~5 CV（柱体积），流速100~300 cm/h。
(3) 用0.2~0.8 CV 的金属离子溶液通过色谱柱，流速50~100 cm/h，金属离子过量50%时足够螯合完全。螯合完全后色谱柱底部的颜色从白色变为螯合特定金属离子的颜色。
(4) 用纯水、0.3 M NaCl 溶液、纯水清洗色谱柱除去过量的金属离子，每步至少5 CV，流速100~300 cm/h。
在装柱前螯合金属离子的过程与上述在色谱柱中螯合一致，可在旋匀仪、反应瓶（釜）中螯合金属离子，利用过滤器清洗螯合前后的填料。
7、在位清洗
通常上述的再生过程可将填料彻底清洗。若发生柱压升高，或为了避免交叉污染，将金属离子剥离后可进行在位清洗过程。可采用以下溶剂进行在位清洗：2 mol/L NaCl，1 mol/L NaOH、70%乙醇或30%异丙醇等。在位清洗可有效去除介质上的杂质，反向效果更佳。清洗后需用3~5 柱体积的纯水除去上述溶剂。
CIP 过程 | 目的 | 2 mol/L NaCl，0.5~2 CV；水，3~5 CV。 | 除去通过离子相互作用结合的杂质 | 1 mol/L NaOH，1~2 h 的接触时间，若除去内毒素则需更长时间；缓冲液A，5~10 CV；水，3~5 CV。 | 去除蛋白沉淀、疏水性蛋白或去除热源等其他杂质 | 30%异丙醇或70%乙醇，5~10 CV；水，3~5 CV。 | 去除紧密结合的脂蛋白和脂质体等 |
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8、填料保存
填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇保存。保存温度在2~8 ℃为宜，不可冻存。
This product is a metal chelating resin functionalized with carboxymethyl aspartate (CM-Asp) groups chelated with cobalt ions, primarily used for affinity purification of His-tagged proteins. Compared with the tetradentate chelator nitrilotriacetic acid (NTA), the most prominent feature of CM-Asp chelated with cobalt ions is its high selectivity. Combined with optimization of the ligand coupling process, this product achieves a balance between binding affinity and specificity: it provides a binding capacity of up to 20 mg/mL for most His-tagged proteins, while the dynamic binding capacity at 10% breakthrough (DBC<sub>10%</sub>) for recombinant protein G and streptavidin with His-tags reaches 25 and 35 mg/mL, respectively. Purity after one-step purification can exceed 90%.
Taking into account both the ligand and the bead matrix, the Cobalt CM-Asp Affinity Resin (His-tag) is suitable for process development and scale‑up production of samples requiring high purity or where Ni<sup>2+</sup>‑chelated resins fail to meet purity requirements.
Aladdin’s Cobalt CM-Asp Affinity Resin is supplied in 20% ethanol with a gel:preservative solution volume ratio of 1:1. The product specification refers to the actual settled gel volume.
| Parameter | Specification |
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| Matrix | High‑rigidity agarose | | Ligand | Carboxymethyl aspartate, pre‑chelated with ~17 μmol Co<sup>2+</sup>/ml gel | | Particle size range (¹) | 30–100 μm | | Mean particle size | ~60 μm | | Dynamic binding capacity (²) | ~20 mg/mL His‑tagged protein | | Recommended linear flow rate | 60–300 cm/h | | Maximum linear flow rate & pressure | 1500 cm/h, 0.5 MPa | | Operating pH | 3–12 (recommended working pH); 3–12 (long‑term stability); 2–14 (short‑term stability, ⁴) | | Chemical stability | Stable in common aqueous buffers, 8 M urea, 6 M guanidine HCl, 70% ethanol, 10 mM β‑mercaptoethanol, etc. Prolonged exposure to reducing agents such as β‑mercaptoethanol is not recommended. | | Storage | 20% ethanol, 2–8 °C | | Shelf life | 5 years |
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Notes
¹ >90% of beads are within the stated range.
² DBC<sub>10%</sub> measured at 10% breakthrough using E. coli‑expressed His‑tagged recombinant protein, 6 min residence time.
³ Maximum tested flow rate with a 10 cm bed height.
⁴ pH stability during CIP after metal ion stripping.
Instruction Manual
1. Column Packing
The following describes column packing procedures when connected to a chromatography system.
(1) All materials should be equilibrated to the operating temperature; degassing of liquids is recommended.
(2) Resin amount calculation: Determine the settled resin volume from the storage suspension. Required settled resin volume = column volume × compression ratio (also called compression factor). Settled volume is the stable volume read after natural settling of the resin in 20% ethanol. The compression ratio for Cobalt CM‑Asp Affinity Resin is ~1.10. Compression can be achieved by lowering the column adaptor or by high flow rate packing.
(3) Resin washing: Shake the resin suspension well, measure the required volume, remove the supernatant, and wash with ~3 resin volumes of purified water. Repeat three times to remove the preservative.
(4) Slurry preparation: Transfer the washed resin to a suitable container, add packing buffer to make a 50–75 v% slurry (the original package contains 50% settled resin). Mix well before use.
(5) Pre‑packing preparation: Add packing buffer to the clean column to remove air from the bottom frit and the lower part of the column, leaving a small amount of distilled water inside. Tighten the bottom cap and align the column vertically.
(6) Packing: Pour the homogeneous slurry slowly into the column (use a packing reservoir if necessary). To avoid introducing air, let the slurry flow down the inner wall. After all resin is added, fill the reservoir with packing buffer, screw on the top cap, and connect the column to the chromatography system.
(7) Compression: Allow the resin to settle by gravity or at a low flow rate (50–150 cm/h). After complete settling (clear interface between resin and liquid), remove the reservoir, attach the top adaptor, and lower the adaptor to the interface. Continue packing at a high flow rate (see recommended table, ensuring back pressure <0.3 MPa) until the interface is clear and stable. Mark the bed height at stable interface. Stop the pump, open the top valve/plug, close the bottom valve/plug, lower the adaptor to the position corresponding to the compression ratio (below the marked height), tighten the adaptor. Packing is complete. After packing, equilibrate the bed at high flow rate.
| Parameter | Cobalt CM‑Asp Affinity Resin |
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| Compression ratio | ~1.10 | | Packing flow rate | ~600 cm/h |
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2. Column Efficiency Test and Evaluation
After packing and before use, column packing quality can be assessed by efficiency tests. Column efficiency is typically evaluated by Height Equivalent to a Theoretical Plate (HETP) and asymmetry factor (As). Acetone or NaCl can be used for efficiency testing – prepare sample and mobile phase according to the table below.
| Sample | 1.0% acetone | 0.8–1.0 M NaCl |
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| Sample volume | 1.0% of column volume | 1.0% of column volume | | Mobile phase | Purified water | 0.4 M NaCl | | Flow rate | 30 cm/h | 30 cm/h | | Detector | UV‑280 nm | Conductivity |
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Calculate HETP, theoretical plate number (N), and asymmetry factor (As) from the UV or conductivity peak:
HETP = L / N
N = 5.54 × (VR / Wh)²
As = a / b
Where: L = bed height; V<sub>R</sub> = retention volume; W<sub>h</sub> = peak width at half height; a = first half‑width at 10% peak height; b = second half‑width at 10% peak height.
Generally, HETP should be less than three times the mean particle size (HETP/D50 < 3, where D50 is the mean particle diameter), and As should be between 0.8 and 1.5.
3. Equilibration and Loading
Metal chelating resins are compatible with phosphate, Tris‑HCl, and other common buffers. A typical buffer system is Tris‑HCl or phosphate buffer (PB) with 150–500 mM NaCl, pH 7.4. The initial buffer is called equilibration buffer (Buffer A). For Cobalt CM‑Asp Affinity Resin, Buffer A usually contains no imidazole, with a pH mostly between 7 and 8 (weakly alkaline).
Before loading, equilibrate the column with Buffer A at the operating flow rate. This usually requires 5–10 column volumes (CV) until conductivity, pH, and other signals are constant and match Buffer A.
Load amount can be defined as “mg target protein / mL resin”, typically 50–80% of the DBC<sub>10%</sub>. Alternatively, determine the load by checking the flow‑through for target protein.
After loading, re‑equilibrate with 3–10 CV of Buffer A.
4. Wash
Add 5–40 mM imidazole to Buffer A to wash away non‑specifically bound impurities. Higher imidazole concentrations remove more impurities but may reduce target protein recovery. The optimal wash imidazole concentration is protein‑dependent; it can be quickly optimized using step elution (e.g., 5, 10, 20 mM imidazole). Wash volume is typically 3–10 CV.
5. Elution
The most common elution method is increasing the imidazole concentration in the elution buffer (imidazole added to Buffer A). Step or linear gradients from 0 to 500 mM imidazole are recommended. For most His‑tagged proteins, 200 mM imidazole elutes the target completely. In some cases, changing pH (not below 4) can also be used.
6. Regeneration
After 5–7 uses or more, regeneration is recommended. Strip metal ions using 0.5–1 CV of 200 mM EDTA + 500 mM NaCl, pH 7. Then chelate with 50–200 mM cobalt chloride solution, and remove excess metal ions with purified water.
Re‑chelation of metal ions – refer to the steps below:
(1) Prepare 50–200 mM cobalt chloride solution. Note solubility under different pH; too high pH may cause precipitation.
(2) Wash the column with 3–5 CV of purified water at 100–300 cm/h.
(3) Pass 0.2–0.8 CV of metal ion solution through the column at 50–100 cm/h. A 50% excess of metal ions is sufficient for complete chelation. After chelation, the bottom of the column changes from white to the characteristic color of the chelated metal ions.
(4) Wash away excess metal ions with purified water, 0.3 M NaCl, and purified water again – each step at least 5 CV at 100–300 cm/h.
Chelation before packing follows the same procedure, which can be carried out in a mixing vessel/reactor, using a filter to wash the resin before and after chelation.
7. Clean‑in‑Place (CIP)
Typically, the regeneration described above cleans the resin thoroughly. If pressure increases or cross‑contamination is to be avoided, perform CIP after metal ion stripping. The following solutions can be used: 2 M NaCl, 1 M NaOH, 70% ethanol, or 30% isopropanol. CIP effectively removes contaminants; backflow direction is more efficient. After CIP, wash with 3–5 CV of purified water to remove the cleaning agents.
| CIP procedure | Purpose |
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| 2 M NaCl, 0.5–2 CV; water, 3–5 CV | Remove ionically bound impurities | | 1 M NaOH, contact time 1–2 h (longer for endotoxin removal); Buffer A, 5–10 CV; water, 3–5 CV | Remove protein precipitates, hydrophobic proteins, pyrogens | | 30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV | Remove tightly bound lipoproteins and lipids |
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8. Resin Storage
The initial storage solution is 20% ethanol. After use, the resin can be stored in 20% ethanol at 2–8 °C. Do not freeze.
ZJ26F0636553
危险品化学品经营许可证（带存储）