Ni-IDA亲和层析介质 6FF (His tag) 是将 IDA 配基通过 9 个原子的间隔臂以醚键固定在高刚性琼脂糖基质上，然后螯合上金属离子 Ni2+而制备成的一种介质。本品以脂糖凝胶为基础微球，FF 为 Fast Flow 的缩写，意为高流速，同时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大于 40 mg/mL 的同时，一步纯化后样品纯度可达 90%以上。
Ni-IDA亲和层析介质 6FF (His tag) 是含亚氨基二乙酸（iminodiacetic acid，IDA）基团的金属螯合填料，适用于大部分带组氨酸标签（His-tag）蛋白的亲和层析。
本品以琼脂糖凝胶为基础微球，FF 为 Fast Flow 的缩写，意为高流速。通过独特的配基修饰工艺，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大于 40 mg/mL 的同时，一步纯化后样品纯度可达 90%以上。
阿拉丁 Ni-IDA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 | 二氨基二乙酸， ~30 μmol Ni²⁺/mL | 粒径范围 ① | 45~165 μm | 平均粒径 | ~90 μm | 动态结合载量② | ≥40 mg/mL 带组氨酸标签蛋白 | 推荐工作流速③ | 60~300 cm/h | 最大流速与压力 ④ | 1500 cm/h，0.5 MPa | 使用pH | 4~8.5（推荐的工作 pH），3~12（长期稳定）；2~14（短期稳定） | 化学稳定性 | 在以下溶液中稳定：常用的水相缓冲液、1 mol/L 氢氧化钠、8 mol/L 尿素、6 mol/L 盐酸胍、70%乙醇等。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
|
注：（有上下标）
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 酸标签的重组金黄色葡萄球菌蛋白 A，6 min 停留时间；
③ 本品可承受高流速和高背压，推荐流速是指该流速可满足大部分柱高下 2~6 min 停留时 间的使用条件；
④10 cm 柱高下的最大测试流速。
金属螯合亲和层析是基于蛋白质分子表面的组氨酸的咪唑基、半胱氨酸的巯基和色氨酸吲哚基能与 Cu²⁺、Ni²⁺、Zn²⁺、Co²⁺等金属离子形成稳定的配位结合而进行生物大分子分离纯化的过程。其中，以 Ni²⁺为螯合离子来纯化 6 个或更多组氨酸组成的 His-tag 标签蛋白最为常见
琼脂糖凝胶修饰上 IDA 配基后，该配体拥有三个配位基团，其中一个氮原子和两个氧原子可以与 Ni²⁺形成牢固的螯合物。Ni²⁺可形成 6 配位数的八面体结构，剩余的三个自由配位点可以与溶剂分子或蛋白质分子结合。Ni²⁺与组氨酸标签蛋白的组氨酸残基的咪唑基团配位结合的过程即为金属螯合填料与目标蛋白的特异性结合过程。提高缓冲液中咪唑的浓度可以将目标蛋白洗脱，洗脱过程中咪唑和目标蛋白竞争与 Ni²⁺的结合。降低 pH 会影响金属离子与配体的结合，因此改变 pH 也是金属螯合层析洗脱过程的方式之一，但 pH 不宜低于 4，pH 过低会将剥离金属离子。通常也用 pH 4 的醋酸钠缓冲液清洗螯合 Ni²⁺后的填料，以除去未稳定螯合的 Ni²⁺。本品IDA 配基与 Ni²⁺形成的三配位螯合物完整度高，从而实现亲和力与特异性的平衡，纯化过程中金属离子脱落少。
金属离子亲和层析过程的非特异性吸附可能来自离子相互作用、金属离子与含组氨酸、半胱氨酸、色氨酸的杂蛋白之间的相互作用。前者通过提高缓冲液的离子强度得到抑制，通常添加 500 mM 的 NaCl；后者可在上样缓冲液中添加少量的咪唑（例如～5 mM）或者在冲洗过程中添加咪唑而除去。经过优化，可以得到样品纯度和收率均较高的结果。当填料性能明显下降后可以用 EDTA 将原金属离子剥离，重新螯合镍后再生使用。不同金属离子对目标蛋白的亲和力与特异性不同，Cu²⁺、Ni²⁺、Zn²⁺、Co²⁺的亲和力依次降低（影响产率），特异性依次升高（影响纯度），可根据分离需求选定合适的金属离子。
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。Ni-IDA亲和层析介质 6FF (His tag) 的压缩比为~1.15。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，抽滤除去液体，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，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 | Ni-IDA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：(公式有上下标）
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（Equilibration & Loading）
本产品与磷酸盐、Tris-HCl 等缓冲体系兼容，其中“20 mM PB+500 mM NaCl（pH=7.4）”的缓冲体系最为常用。 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。
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。上样前需要对样品进行离心（10000 g 以上）、过滤（0.22 或 0.45 μm）处理，以免堵塞色谱柱。
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 （Wash）
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（Elution）
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，结合较 弱的蛋白可在 pH 6 时可洗脱，结合较强的蛋白可在 pH 6~4 时洗脱，需注意 pH 不能低 于 4。
6、再生（Regeneration）
在多次使用后，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 的金属盐溶液与 IDA 配 基螯合，用纯水将过量的金属离子除去，用 20 mM 醋酸钠+0.5 M NaCl（pH=4.0）缓冲 液将螯合不稳定的金属离子除去。
7. 在位清洗（CIP）
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 |
|
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。
Ni-IDA Affinity Resin (His tag) is prepared by immobilizing the IDA ligand onto a highly rigid agarose matrix via a 9-atom spacer arm through an ether bond, followed by chelation with Ni²⁺ metal ions. This product is based on agarose microspheres, and "FF" stands for Fast Flow, indicating high flow rates. It achieves a balance between affinity and specificity: binding capacity for most His-tagged proteins is >40 mg/mL, while purity after a single purification step can reach >90%.
Ni-IDA Affinity Resin (His tag) is a metal chelating resin containing iminodiacetic acid (IDA) groups, suitable for affinity purification of most proteins with a polyhistidine tag (His-tag).
This product is based on agarose microspheres, with "FF" standing for Fast Flow, indicating high flow rates. Through a unique ligand modification process, it achieves a balance between affinity and specificity: binding capacity for most His-tagged proteins is >40 mg/mL, while purity after a single purification step can reach >90%.
Aladdin's Ni-IDA Affinity Resin (His tag) is stored in 20% ethanol, with a gel-to-preservative solution volume ratio of 1:1. Our product specifications are based on the actual gel volume.
Parameter | Specification | Matrix | 6% cross-linked agarose | Ligand | Iminodiacetic acid, ~30 µmol Ni²⁺/mL | Particle Size Range ① | 45~165 µm | Mean Particle Size | ~90 µm | Dynamic Binding Capacity ② | ≥40 mg/mL His-tagged protein | Recommended Working Flow Rate ③ | 60~300 cm/h | Maximum Flow Rate & Pressure ④ | 1500 cm/h, 0.5 MPa | Operating pH | 4~8.5 (recommended working pH); 3~12 (long-term stability); 2~14 (short-term stability) | Chemical Stability | Stable in: common aqueous buffers, 1 mol/L NaOH, 8 mol/L urea, 6 mol/L guanidine hydrochloride, 70% ethanol, etc. | Storage | 20% ethanol, 2~8℃ | Shelf Life | 5 years |
|
Notes:（有上下标）
① >90% of microspheres are within this size range.
② DBC10% test conditions: 10% breakthrough, E. coli-expressed His-tagged recombinant Staphylococcus aureus protein A, 6 min residence time.
③ This product can withstand high flow rates and high backpressure. The recommended flow rate meets the conditions for a 2~6 min residence time for most column heights.
④ Maximum test flow rate at 10 cm column height.
Metal chelate affinity chromatography is based on the stable coordination binding between metal ions (Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺) and imidazole groups of histidine, thiol groups of cysteine, and indole groups of tryptophan on the surface of protein molecules. Among these, the use of Ni²⁺ as the chelated metal ion to purify proteins with a His-tag (consisting of 6 or more histidines) is most common.
After modification of agarose with the IDA ligand, the ligand possesses three coordinating groups (one nitrogen atom and two oxygen atoms) that can form a stable chelate with Ni²⁺. Ni²⁺ can form an octahedral structure with a coordination number of 6. The remaining three free coordination sites can bind to solvent molecules or protein molecules. The coordination binding of Ni²⁺ to the imidazole groups of histidine residues on the His-tagged protein constitutes the specific binding process between the metal chelating resin and the target protein. Increasing the imidazole concentration in the buffer elutes the target protein, as imidazole competes with the target protein for binding to Ni²⁺. Lowering the pH affects the binding between the metal ion and the ligand, thus pH changes are also a method for elution in metal chelate chromatography. However, the pH should not be lower than 4, as excessively low pH will strip the metal ions. Typically, pH 4 sodium acetate buffer is used to wash the Ni²⁺-chelated resin to remove unstably chelated Ni²⁺. The IDA ligand in this product forms a highly stable tridentate chelate with Ni²⁺, achieving a balance between affinity and specificity with minimal metal ion leaching during purification.
Non-specific adsorption in metal ion affinity chromatography may arise from ionic interactions or interactions between metal ions and contaminating proteins containing histidine, cysteine, or tryptophan. The former is suppressed by increasing the ionic strength of the buffer, typically by adding 500 mM NaCl. The latter can be reduced by adding a small amount of imidazole (e.g., ~5 mM) to the loading buffer or during the wash step. Through optimization, both high purity and high yield can be achieved. When resin performance significantly declines, EDTA can be used to strip the metal ions, and the resin can be regenerated by re-chelating with nickel. Different metal ions have varying affinities and specificities for target proteins. Affinity (influencing yield) decreases in the order Cu²⁺ > Ni²⁺ > Zn²⁺ > Co²⁺, while specificity (influencing purity) increases. The appropriate metal ion can be selected based on purification requirements.
Instructions for Use
1. Column Packing
The following describes the column packing method for the resin when connected to a chromatography system.
(1) All materials used should be at the same temperature as the chromatography operation. Liquids should preferably be degassed.
(2) Resin Volume Calculation: Determine the resin volume by sedimentation. Required settled resin volume = Column Volume × Compression Ratio (also called compression factor). Settled volume refers to the stable volume read after the resin has completely settled in the 20% ethanol storage solution. The compression ratio for Ni-IDA Affinity Chromatography Medium 6FF (His-tag) is ~1.15. To achieve this compression ratio, the column head can be lowered, or high flow rates can be used to pack the column.
(3) Resin Washing: Thoroughly resuspend the resin slurry, measure the required volume, and vacuum filter to remove the liquid. Wash the resin three times with approximately 3 volumes of purified water each time to remove the storage solution.
(4) Packing Slurry Preparation: Transfer the washed resin to a suitable container, add an appropriate amount of packing buffer to make a 50-75 v% slurry. Mix well before use.
(5) Pre-packing Preparation: Add packing buffer to the bottom of the clean chromatography column to remove air from the bottom frit and the lower part of the column. Leave a small amount of distilled water in the column, tighten the bottom cap, and adjust the column vertically.
(6) Packing: Pour the well-mixed resin slurry slowly into the chromatography column at once (use a packing reservoir if necessary). To avoid introducing air bubbles, allow the slurry to flow down along the inner wall of the column. After all the resin is added, fill the packing reservoir with packing buffer, tighten the reservoir cap, and connect the column to the chromatography system.
(7) Compression: Allow the resin in the column to settle naturally (or at a low flow rate of 50-150 cm/h). Once the resin is completely settled (a clear interface between the resin and the liquid is visible), remove the packing reservoir, attach the top adaptor, and lower the adaptor to the interface. Continue to compress the resin using a high flow rate (see recommended flow rates below, ensuring column backpressure does not exceed 0.3 MPa) until the interface is clear and stable. Mark the column height at the stable interface. Stop the pump. Open the valve/plug on the top adaptor, close the valve/plug at the column bottom, lower the adaptor to the position corresponding to the compression ratio below the marked position, and tighten the adaptor. Packing is complete. After packing, equilibrate the resin in the column using a high flow rate.
Packing Condition | Ni-IDA Affinity Resin (His tag) | Compression ratio | 1.15 | Packing flow rate | 600 cm/h |
|
2. Column Efficiency Test & Evaluation
Column efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.
Item | 1.0% Acetone | 0.8–1.0 M NaCl | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
|
Calculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:（公式有上下标）
- HETP = L / N
- N = 5.54 × (Vr / Wh)2
- As = a / b
Where:L = column height; Vr = 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, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration & Loading
The product is compatible with buffer systems such as phosphate and Tris-HCl. The buffer system "20 mM PB + 500 mM NaCl, pH 7.4" is most commonly used. The initial buffer is called the equilibration buffer, designated as Buffer A. Buffer A is characterized by containing no or low amounts of imidazole and having a pH typically between 7 and 8 (weakly alkaline). In practice, if the sample load volume is very large, to reduce costs, imidazole and NaCl may not be added to the sample to suppress non-specific adsorption; instead, impurities can be removed by washing after loading.
Before loading, equilibrate the column with Buffer A at the operating flow rate. This process typically requires 5-10 column volumes of Buffer A until parameters such as conductivity and pH are constant and correspond to Buffer A.
The loading amount can be set as "mg target protein / mL resin," typically 50-80% of the DBC10%. Besides DBC10%, the target protein in the flow-through can also be tested to determine the loading amount. Samples must be centrifuged (≥10,000 g) and filtered (0.22 or 0.45 µm) before loading to prevent column clogging.
After loading, re-equilibrate the column with 3-10 column volumes of Buffer A.
4. Wash
Add 5-40 mM imidazole to Buffer A to wash the column and remove non-specifically bound contaminating proteins. Higher imidazole concentrations remove contaminants more thoroughly but may reduce the yield of the target protein. The optimal imidazole concentration for the wash step depends on the specific protein properties. Wash conditions can be quickly optimized using step elution washes (e.g., 5 mM, 10 mM, 20 mM...). The wash volume is typically 3-10 column volumes.
5. Elution
The most common elution method is to increase the imidazole concentration in the elution buffer (Buffer A with added imidazole). Step elution or linear gradient elution within the range of 0-500 mM imidazole is recommended. For most His-tagged proteins, 200 mM imidazole is sufficient for complete elution. In some cases, pH changes can also be used. Weakly bound proteins can be eluted at pH 6, while more strongly bound proteins can be eluted at pH 6-4. Note that the pH should not be lower than 4.
6. Regeneration
After multiple uses, the resin needs to be regenerated. Metal ions can be stripped using 0.5-1 column volume of 200 mM EDTA + 500 mM NaCl, pH 7 buffer. Then, re-chelating with a 50-200 mM metal salt solution, followed by washing with purified water to remove excess metal ions, and finally washing with 20 mM sodium acetate + 0.5 M NaCl, pH 4.0 buffer to remove unstably chelated metal ions.
7. Clean-in-Place (CIP)
Typically, the regeneration process described above cleans the resin thoroughly. If column backpressure increases, or to avoid cross-contamination, a CIP procedure can be performed after stripping the metal ions. The following solutions can be used for CIP: 2 mol/L NaCl, 1 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP effectively removes impurities from the resin; backflow is more effective. After CIP, wash with 3-5 column volumes of purified water to remove the CIP solutions.
CIP Procedure | Purpose | 2 mol/L NaCl, 0.5–2 CV; water, 3–5 CV | Removes impurities bound by ionic interactions | 1 mol/L NaOH, 1–2 h contact time (longer for endotoxin removal); buffer A, 5–10 CV; water, 3–5 CV | Removes protein precipitates, hydrophobic proteins, or other impurities such as pyrogens | 30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV | Removes tightly bound lipoproteins, liposomes, etc. |
|
8. Resin Storage
The initial storage solution for the resin is 20% ethanol. After use, it can still be stored in 20% ethanol. The suitable storage temperature is 2–8 °C; do not freeze.
ZJ26F0939305
危险品化学品经营许可证（带存储）