Ni-TED 亲和层析介质 6FF (His tag) 是金属离子脱落率最低的一款金属螯合亲和填料，配基为三羧甲基乙二胺（tris-carboxymethyl ethylene diamine，TED）基团，配位金属离子为Ni²⁺，FF为Fast Flow的缩写。本产品的TED配基与Ni²⁺形成的五配位结构稳定性好，对螯合剂、还原剂、酸、碱 等试剂有着出色的耐受性，可在10 mM EDTA、100 mM β-ME或50 mM DTT等条件下正常使用，可在pH=1或pH=14的条件下处理24h后保持出色的色谱性能，也长期保存在10 mM NaOH 中，洗脱液中需要的咪唑浓度相对其他金属螯合填料更低。
因此，Ni-TED 亲和层析介质 6FF (His tag) 适合用于下列纯化体系：
1. 目标蛋白中含半胱氨酸或者对氧敏感，需要添加β-ME或DTT时；
2. 目标蛋白中含半胱氨酸且有金属反应性，需要添加DTT和EDTA时；
3. 目标蛋白容易被金属蛋白酶水解，需要添加EDTA时；
4. 目标蛋白对高浓度咪唑敏感，需要降低洗脱液中咪唑浓度时；
5. 真核细胞表达体系中本身含β-ME或EDTA的情况；
6. 对产物中金属离子含量要求严格时。
Ni-TED 亲和层析介质 6FF的配基偶联工艺经过了特殊的设计，使得成品的压力/流速特性得到大幅提升，最大流速和耐压分别超过1500 cm/h和0.5 MPa。通过配基修饰过程的优化，本产品在亲和力（载量）和特异性（选择性）之间取得良好的均衡：对带组氨酸标签蛋白的结合量大于20 mg/mL的同时，一步纯化后样品纯度可达90%以上。
阿拉丁Ni-TED 亲和层析介质 6FF (His tag) 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
| 6%交联琼脂糖
|
配基 ①
| 三羧甲基乙二胺，螯合~60 μmol Ni²⁺/mL
|
粒径范围 ②
| 45~165 μm
|
平均粒径
| ~90 μm
|
动态结合载量 ③
| ≥20 mg/mL带组氨酸标签蛋白
|
推荐工作流速 ④
| 60~300 cm/h
|
最大流速与压力 ⑤
| 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）
|
化学稳定性
| 1) 纯化缓冲液：常用的水相缓冲液，可含有10 mM EDTA，100 mM β-ME 或50 mM DTT。
2) 在位清洗（CIP）处理：1 M NaOH、1~2 M NaCl、70%乙醇、30%异丙醇，0.1~0.5%非离子型表面活性剂。
3) 在下述溶液中处理特定时间，填料性能保持稳定：10 mM NaOH（长期）、0.1 M盐酸（24 h）、6 M盐酸胍（24 h）。
|
储存
| 20%乙醇，2~8℃
|
保质期
| 5年 |
注：
① 三羧甲基乙二胺（TED）基团与基球之间有十个原子长度的间隔臂，为亲和填料提供更加高效的捕获效果。
② 90%体积以上的微球在此粒径范围。
③ 10%穿透下的动态结合载量（DBC10%），测试的样品为大肠杆菌表达带组氨酸标签的重组蛋白，6 min停留时间。
④ 并非指只能在该流速范围内工作。需结合柱高确定适宜的工作流速，通常情况下上样的停留时 间 2~6 min 范围内可保持出色的亲和捕获能力和纯化效率。CIP 过程的流速则一般不超过150 cm/h。
⑤ 10 cm柱高下的最大测试流速及该最大流速下的压力。
金属螯合亲和层析介绍
金属离子亲和层析基于蛋白质分子表面的组氨酸咪唑基、半胱氨酸巯基和色氨酸吲哚基，能与 Ni²⁺、Co²⁺、Cu²⁺、Zn²⁺等形成配位结合实现生物大分子分离纯化；其中以 Ni²⁺为螯合离子纯化带 6 个以上组氨酸标签蛋白的应用最为常见，本说明书也以该体系为例简要阐述Ni-TED 亲和层析介质 6FF (His tag) 的亲和原理（图1）。
基球修饰 TED 配基后，该配体拥有五个配位基团，其中两个氮原子和三个氧原子与 Ni²⁺形成牢固螯合物；Ni²⁺可形成 6 配位数的八面体结构，剩余一个自由配位点可与溶剂分子或蛋白质分子结合。Ni²⁺与组氨酸标签蛋白的咪唑基团配位结合，即为填料与目标蛋白的特异性结合过程；提高缓冲液中咪唑浓度，可通过竞争结合方式洗脱目标蛋白。
非特异性吸附主要源于离子相互作用、金属离子与含组氨酸 / 半胱氨酸 / 色氨酸蛋白的相互作用：前者可通过提升缓冲液离子强度（添加 500 mM NaCl）抑制；后者可在上样缓冲液中添加少量咪唑（~5 mM）或冲洗过程中加咪唑除去。
图1：Ni-TED 亲和层析介质 6FF (His tag) 的纯化原理示意图
使用方法参考
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）所有需用材料的温度需与色谱操作温度一致，液体优先做脱气处理。
（2）填料用量计算：沉降填料体积 = 柱体积 × 压缩比（Ni-TED 亲和层析介质 6FF (His tag) 压缩比为 1.15），沉降体积为填料在 20% 乙醇保存液中自然沉降完全后的稳定体积；可通过柱头下压或高流速压柱的方式达到压缩比。
（3）填料清洗：充分摇匀填料悬液后量取体积，抽去液体，用约 3 倍填料体积的纯化水洗涤，重复 3 次，去除保存液。
（4）填料悬液准备：将填料转移至合适容器，加入装柱液制成 50~75 v% 的悬液（原包装填料体积分数为 67%），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，除去下垫片及柱底空气，柱内保留少量蒸馏水，拧紧下堵头，使柱子垂直于地面。
（6）装填：将搅匀的填料悬液一次性缓慢沿层析柱内壁倒入（必要时使用装柱器），避免引入气泡；填料加完后，用装柱液加满柱子，拧紧上盖，连接层析系统。
（7）压柱：使柱内填料自然沉降（或 50~150 cm/h 低流速下沉降），待界面清晰稳定后，移除装柱器，装上柱头并下降至界面处；以高流速（柱压不超过 0.3 MPa）继续压柱至界面清晰稳定，标记界面稳定柱高；停泵后，下压柱头至标记位置下方与压缩比对应位置，旋紧柱头，完成装柱。装柱完成后，用高流速平衡柱内填料。
装柱条件
| Ni-TED 亲和层析介质 6FF (His tag)
|
压缩比
| 1.15
|
装柱流速
| 600 cm/h |
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
|
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 × (Vʀ / Wₕ)²
As = a / b
其中：L为柱高； Vʀ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
（1）金属螯合填料与磷酸盐、Tris-HCl 等缓冲体系兼容，其中 20 mM PB+500 mM NaCl （pH=7.4）的体系最为常用。初始的缓冲液称为平衡缓冲液，记为缓冲液A。缓冲液A的特点为不含或者含少量咪唑，pH在7~8之间居多，呈弱碱性。实际使用过程中，如 果上样料液的体积特别大，从降低成本的角度考虑，可不往料液中添加咪唑和NaCl来 抑制非特异性吸附，而是在上样后进行冲洗来去除杂质。
（2）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。
（3）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 咪唑冲洗层析柱，可除去非特异性结合的杂蛋白；咪唑浓度越高，杂蛋白去除越彻底，但会降低目标蛋白收率。可通过阶跃洗杂（如 5 mM、10 mM、20 mM……）快速优化条件，洗杂体积通常为 3~10 个柱体积。
5、洗脱
最常用洗脱方式为提高洗脱液中咪唑浓度（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线性梯度洗脱以确定最优条件；多数带组氨酸标签蛋白，50~150 mM 咪唑 浓度即可实现目标蛋白完全洗脱。
6、在位清洗（CIP）
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 M 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 M 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 |
7. 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2~30℃为宜，不可冻存。
Ni-TED Affinity Resin 6FF (His tag) is a metal chelate affinity resin with the lowest metal ion leaching rate. The ligand is a tris-carboxymethyl ethylene diamine (TED) group, the coordinated metal ion is Ni²⁺, and FF stands for Fast Flow. The penta-coordinate structure formed between the TED ligand and Ni²⁺ in this product exhibits excellent stability, offering outstanding tolerance to chelating agents, reducing agents, acids, alkalis, and other reagents. It can be used under conditions such as 10 mM EDTA, 100 mM β-ME, or 50 mM DTT, maintains excellent chromatographic performance after 24 hours of treatment at pH 1 or pH 14, and can also be stored long-term in 10 mM NaOH. The imidazole concentration required in the elution buffer is lower than that needed for other metal chelate resins.
Therefore, Ni-TED Affinity Resin 6FF (His tag) is suitable for the following purification systems:
When the target protein contains cysteine or is oxygen-sensitive and requires the addition of β-ME or DTT;
When the target protein contains cysteine and exhibits metal reactivity, requiring the addition of DTT and EDTA;
When the target protein is susceptible to hydrolysis by metal proteases, requiring the addition of EDTA;
When the target protein is sensitive to high concentrations of imidazole, requiring a reduction in imidazole concentration in the elution buffer;
When the eukaryotic expression system itself contains β-ME or EDTA;
When strict requirements exist for the metal ion content in the final product.
The ligand coupling process of Ni-TED Affinity Resin 6FF has been specially designed to significantly enhance the pressure/flow rate characteristics of the final product, with maximum flow rate and pressure resistance exceeding 1500 cm/h and 0.5 MPa, respectively. Through optimization of the ligand modification process, this product achieves a good balance between affinity (capacity) and specificity (selectivity): while the binding capacity for histidine-tagged proteins exceeds 20 mg/mL, the sample purity after single-step purification can reach over 90%.
Aladdin Ni-TED Affinity Resin 6FF (His tag) is stored in 20% ethanol, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
|
Matrix
| 6% cross-linked agarose
|
Ligand ①
| Tris-carboxymethyl ethylene diamine, chelating ~60 μmol Ni²⁺/mL
|
Particle size range ②
| 45~165 μm
|
Mean particle size
| ~90 μm
|
Dynamic binding capacity ③
| ≥20 mg/mL histidine-tagged protein
|
Recommended operating flow rate ④
| 60~300 cm/h
|
Maximum flow rate and pressure ⑤
| 1500 cm/h，0.5 MPa
|
Operating pH
| 3–12 (recommended working pH), 3–12 (long-term stability); 2–14 (short-term stability)
|
Chemical stability
| 1) Purification buffers: common aqueous buffers, may contain 10 mM EDTA, 100 mM β-ME, or 50 mM DTT.
2) CIP treatments: 1 M NaOH, 1–2 M NaCl, 70% ethanol, 30% isopropanol, 0.1–0.5% nonionic surfactants.
3) The resin remains stable after treatment for specific periods in the following solutions: 10 mM NaOH (long-term), 0.1 M HCl (24 h), 6 M guanidine hydrochloride (24 h).
|
Storage
| 20% ethanol, 2–8°C
|
Shelf life
| 5 years |
Notes:
① The tris-carboxymethyl ethylene diamine (TED) group has a ten-atom spacer arm between the ligand and the base bead, providing more efficient capture for the affinity resin.
② More than 90% of the beads are within this particle size range.
③ Dynamic binding capacity at 10% breakthrough (DBC<sub>10%</sub>). Tested with histidine-tagged recombinant protein expressed in E. coli, with a residence time of 6 minutes.
④ Not limited to this flow rate range. The appropriate flow rate should be determined based on column height. Typically, a residence time of 2–6 minutes during sample loading maintains excellent affinity capture capacity and purification efficiency. The flow rate during CIP should generally not exceed 150 cm/h.
⑤ Maximum tested flow rate and corresponding pressure at a column height of 10 cm.
Introduction to Metal Chelate Affinity Chromatography
Metal chelate affinity chromatography is based on the imidazole groups of histidine, thiol groups of cysteine, and indole groups of tryptophan on the surface of protein molecules. These groups can form coordinate bonds with Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, etc., enabling the separation and purification of biomacromolecules. Among these, the purification of proteins with six or more histidine tags using Ni²⁺ as the chelated ion is the most common application. This manual also uses this system as an example to briefly explain the affinity principle of Ni-TED Affinity Resin 6FF (His tag) (Figure 1).
After the base beads are modified with TED ligands, the ligand possesses five coordination groups, with two nitrogen atoms and three oxygen atoms forming a stable chelate with Ni²⁺. Ni²⁺ can form an octahedral structure with a coordination number of 6, with the remaining free coordination site available to bind to solvent molecules or protein molecules. The coordination binding between Ni²⁺ and the imidazole groups of histidine-tagged proteins constitutes the specific binding process between the resin and the target protein. Increasing the imidazole concentration in the buffer allows elution of the target protein through competitive binding.
Non-specific adsorption mainly arises from ionic interactions and interactions between metal ions and histidine/cysteine/tryptophan-containing proteins. The former can be suppressed by increasing the ionic strength of the buffer (by adding 500 mM NaCl); the latter can be addressed by adding a small amount of imidazole (~5 mM) to the loading buffer or by adding imidazole during the wash step.
Figure 1: Schematic diagram of the purification principle of Ni-TED Affinity Resin 6FF (His tag)
Usage Reference
1. Column Packing
The following describes the column packing method when connected to a chromatography system.
(1) The temperature of all required materials should match the chromatography operating temperature, and degassing of liquids is recommended.
(2) Calculation of resin volume: Settled resin volume = column volume × compression ratio (the compression ratio of Ni-TED Affinity Resin 6FF (His tag) is 1.15). Settled volume refers to the stable volume of the resin after complete natural sedimentation in 20% ethanol preservation solution. The compression ratio can be achieved by lowering the column adaptor or by packing at high flow rates.
(3) Resin washing: After thoroughly mixing the resin suspension and measuring the volume, remove the liquid, wash with approximately 3 resin volumes of purified water, repeat 3 times to remove the preservation solution.
(4) Preparation of resin suspension: Transfer the resin to a suitable container, add packing buffer to prepare a 50–75 v% suspension (the original packaging resin volume fraction is 67%), and mix well before use.
(5) Pre-packing preparation: Add packing buffer to the cleaned chromatography column from the bottom, remove air from the bottom frit and column bottom, leave a small amount of distilled water in the column, tighten the bottom cap, and ensure the column is perpendicular to the floor.
(6) Packing: Slowly pour the mixed resin suspension along the inner wall of the chromatography column in one go (use a packing reservoir if necessary), avoiding introduction of air bubbles. After adding the resin, fill the column with packing buffer, tighten the top cap, and connect to the chromatography system.
(7) Compaction: Allow the resin to settle naturally (or at a low flow rate of 50–150 cm/h). After the interface is clear and stable, remove the packing reservoir, install the top adaptor, and lower it to the interface. Continue packing at a high flow rate (column pressure not exceeding 0.3 MPa) until the interface is clear and stable, then mark the stable column height. After stopping the pump, lower the top adaptor to the position below the marked position corresponding to the compression ratio, tighten the adaptor, and complete the packing. After packing, equilibrate the resin in the column at a high flow rate.
Packing Condition
| Ni-TED Affinity Resin 6FF (His tag)
|
Compression Factor
| 1.15
|
Packing Flow Rate
| 600 cm/h |
2. Column Efficiency Determination and Evaluation
After packing and before use, the packing quality of the chromatography column can be verified by column efficiency determination and evaluation. Column efficiency is typically assessed using Height Equivalent to a Theoretical Plate (HETP) and asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as the sample. Prepare the sample solution and mobile phase according to the table below.
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
|
Sample Volume
| 1.0% CV
| 1.0% CV
|
Mobile Phase
| Pure Water
| 0.4 M NaCl
|
Flow Rate
| 30 cm/h
| 30 cm/h
|
Detector
| UV-280 nm
| Conductivity
|
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
Where: L = column height; Vʀ = retention volume; Wₕ = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.
Generally, the HETP value should be less than three times the average particle size of the resin (i.e., HETP/D50 < 3, where D50 is the average particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration and Loading
(1) Metal chelate resins are compatible with buffer systems such as phosphate and Tris-HCl, with the system 20 mM PB + 500 mM NaCl (pH 7.4) being the most commonly used. The initial buffer is called the equilibration buffer, designated as Buffer A. Buffer A is characterized by containing no or low levels of imidazole, with a pH mostly between 7 and 8, being weakly alkaline. In practice, if the sample loading volume is particularly large, to reduce costs, one may choose not to add imidazole and NaCl to the sample solution to suppress non-specific adsorption, but instead remove impurities by washing after loading.
(2) Before loading, equilibrate the chromatography column with Buffer A at the operating flow rate. This process typically requires 5–10 column volumes of Buffer A, and is considered complete when detection signals such as conductivity and pH remain constant and correspond to those of Buffer A.
(3) The loading amount can be set based on "mg target protein/mL resin", typically 50–80% of the DBC<sub>10%</sub>. In addition to DBC<sub>10%</sub>, the loading amount can also be determined by testing the target protein in the flow-through during loading. After loading, re-equilibrate the chromatography column with 3–10 column volumes of Buffer A.
4. Wash
Adding 5–40 mM imidazole to Buffer A to wash the chromatography column removes non-specifically bound impurity proteins. The higher the imidazole concentration, the more thorough the removal of impurity proteins, but this may reduce the yield of the target protein. Conditions can be rapidly optimized through stepwise washing (e.g., 5 mM, 10 mM, 20 mM...). The wash volume is typically 3–10 column volumes.
5. Elution
The most commonly used elution method is increasing the imidazole concentration in the elution buffer (adding imidazole to Buffer A). Stepwise or linear gradient elution in the range of 0–500 mM is recommended to determine optimal conditions. For most histidine-tagged proteins, complete elution of the target protein can be achieved with an imidazole concentration of 50–150 mM.
6. Cleaning-in-Place (CIP)
If column pressure increases or to avoid cross-contamination, the metal ions can be stripped off and a CIP process performed. The following solvents can be used for CIP: 2 mol/L NaCl, 1 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP effectively removes impurities on the resin, with backflushing being more effective. After cleaning, remove the above solvents with 3–5 column volumes of purified water.
CIP Process
| Purpose
|
2 M NaCl, 0.5–2 CV; water, 3–5 CV.
| Removal of impurities bound through ionic interactions.
|
1 M NaOH, contact time of 1–2 hours (longer for endotoxin removal); Buffer A, 5–10 CV; water, 3–5 CV.
| Removal of protein precipitates, hydrophobic proteins, or endotoxins and other impurities.
|
30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV.
| Removal of tightly bound lipoproteins and liposomes. |
7. Resin Storage
The initial preservation solution for the resin is 20% ethanol. After use, it can continue to be stored in 20% ethanol. The storage temperature should be 2–30°C, and freezing should be avoided.
ZJ26F0535128
危险品化学品经营许可证（带存储）