储存缓冲液：20% (v/v) Ethanol
金属螯合亲和层析，又称固定化金属离子亲和层析（Immobilized Metal Ion Affinity Chromatography, IMAC）。利用蛋白质表面的某些氨基酸（如组氨酸、色氨酸、半胱氨酸等）和金属离子Ni2+、Cu2+、Zn2+、Co2+、Ca2+、Fe3+等）
特异性结合的原理，实现蛋白质的分离纯化。
本产品是将次氮基三乙酸（NTA）配基偶联至6%高度交联琼脂糖制备而成的⾦属螯合亲和层析介质（未螯合金属离子），可提供4个配位位点同金属离子螯合，螯合的金属离子更稳定，可耐受⼀定浓度的还原剂。用户螯合金属离子后，可用于生物制药和生物工程下游蛋白质及多肽的分离纯化。
阿拉丁金属螯合-NTA亲和层析介质6FF储存在20%乙醇中，凝胶和保护液的体积比为7:3，我司产品规格为实际凝胶的体积。
参数 | 指标 | 基质 | 6%高度交联琼脂糖 | 功能基团 | 次氮基三乙酸（NTA） | 平均粒径 | 90 μm | 螯合金属能力 | 15 μmol Ni2+/mL 介质 | 结合载量 | 约40 mg 组氨酸标签蛋白/mL 介质（螯合Ni2+后） | 建议流速 | <400 cm/h | 建议压力 | <0.3 MPa（3 bar） | 工作pH | 4-8.5 | pH稳定性 ① | 3-12（长期）；2-14（短期） | 化学稳定性 | 常用缓冲液中稳定；避免使用螯合剂，如EDTA、柠檬酸盐等。 | 保存条件 | 20%乙醇；2-8℃ | 保质期 | 5年 |
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注：
① 该处指在未螯合金属离子时的介质稳定性。长期，指在长时间内保持稳定而不对其后续性能产生不利影响的pH 范围。短期，指根据经验给出的再生、在位清洗和消毒的pH 范围。
使用说明
1、装柱
1.1 装柱缓冲液准备
纯化水，超声脱气15 min。
1.2 层析介质准备
计算所需层析介质的量（压缩系数约为1.15），并称取备用。用真空抽滤瓶置换至装柱缓冲液中。置换的介质加入一定量装柱缓冲液，制成约50%浓度的胶悬液。
1.3 层析柱准备
检查层析柱，确保各部件完整、干净。安装下柱头，拧紧O型圈后，将层析柱垂直固定在铁架台上，用水平仪检查并调整使层析柱保持竖直。用注射器吸取装柱缓冲液，接于柱底的出口处，缓慢推出注射器内液体，排除底部筛网的气泡，取下注射器后拧上堵头。层析柱中加入约2 cm 高的装柱缓冲液。
1.4 装柱（以直径16 mm，床高10 cm 的层析柱为例）
混匀胶悬液，用玻璃棒引流缓慢倒入层析柱中，若液面离柱顶还有空隙，加装柱缓冲液充满。将调节器连接至层析系统，启动泵以一定流速排除管路及顶部筛网中的气泡后暂停。将调节器沿45°放入柱中，固定好调节器并拧紧密封圈，注意避免气泡进入。拧开层析柱底部的堵头，将底部管路放入到废液缸中，流速设为60 cm/h，至介质界面无变化后，设置流速为600 cm/h，并逐步降低流速到300 cm/h，维持该流速45 min，用记号笔在界面处做好标记，暂停系统。拧上底部堵头，层析柱上端与泵断开，稍拧松调节器上的密封圈，将柱头压到胶面以下3 mm 处，拧紧密封圈。将层析柱顶部和底部管路连接到层析系统上，进行柱效测定。
2、柱效测定和评价
装柱完成后应立即检测柱效。通常用每米理论塔板数N/m、峰不对称因子As等术语来衡量层析柱的性能。柱效越高，层析柱分离能力越强。为了更加准确地反映层析柱柱效，检测时待测样品上样体积为柱体积的1.0%，线性流速控制在15-30 cm/h。为了防止样品的稀释，尽量在接近层析柱的入口处上样。
检测方法 | 丙酮法 | NaCl法 | 样品 | 1.0%（v/v）丙酮水溶液 | 0.8 M NaCl 水溶液 | 样品体积 | 1.0%柱体积 | 1.0%柱体积 | 流动相 | 纯水 | 0.4 M NaCl 水溶液 | 流速 | 30 cm/h | 30 cm/h | 检测器 | UV-280 nm | 电导 |
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柱效计算
根据UV曲线（或电导率曲线）计算N/m、和As，公式如下：
- N/m = N/L
- N = 5.54(VR/Wh)2
- As=b/a备注：L为柱高（单位为m）； 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。 As应在0.8~1.8之间。N/m>3000
3、分离纯化
金属螯合-NTA亲和层析介质6FF需先螯合金属离子，才能进行分离纯化，螯合金属离子的详细步骤参见“4. 再生”。
择金属离子：实验中经常使用Cu2+、Zn2+Ni2+，Cu2+和Zn2+最常用作纯化非标签蛋白，Cu2+可以和某些蛋白强力结合，且某些蛋白只与Cu2+结合。Zn2+和某些蛋白的结合较弱且仅能结合少数蛋白，常被用来纯化特定蛋白。Ni2+常用于纯化含有His 标签的蛋白。此外，Co2+、Fe3+和Ca2+也可作为备选。
缓冲液首选磷酸盐缓冲液，pH 中性至弱碱性（pH7.0-8.0），避免使用EDTA 及柠檬酸盐等，Tris-HCl 也可以使用，但在金属离子与蛋白质亲和力很弱的情况下应避免使用。下表列出了常见添加试剂及浓度。为了减少宿主蛋白与介质的非特异吸附，结合缓冲液和样品中通常加入低浓度咪唑（20-40 mM）。缓冲液中必须加入0.15-0.5 M NaCl 以消除离子吸附作用。
不影响蛋白质结合的添加物：
添加物 | 常用浓度 | 添加物 | 常用浓度 | 磷酸盐、Tris、硼酸盐、HEPES | 20-100mM | 非离子型去污剂 | ≤2% | NaCl | ≤2M | Triton X-100 | ≤2% | KCl | ≤1M | Tween-20 | ≤2% | 盐酸胍 | ≤6M | 辛基葡糖苷 | ≤2% | 尿素 | ≤8M | 十二烷基麦芽糖柑 | ≤2% | 甘油 | ≤50% | C12E8、C10E6 | ≤2% | 异丙醇 | ≤60% | PMSF(蛋白酶抑制剂) | 1 mM | 乙醇 | ≤30% | 胃蛋白酶抑制剂 | 1 μM | 1%苯甲脒（蛋白酶抑制剂） | ≤1 mM | 亮抑酶肽(蛋白酶抑制剂) | 0.5 μg/mL |
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可能破坏蛋白质结合的添加物（尽量避免使用）：
添加物 | 常用浓度 | 添加物 | 常用浓度 | β-巯基乙醇 | <20 mM | 柠檬酸盐 | 可耐受低浓度 | 还原谷胱甘肽 | <10 mM | 组氨酸 | 可替代咪唑 | 强还原剂（DTT、TCEP、DTE）① | <5 mM | 甘氨酸 | - | 金属螯合剂 (EDTA、EGTA)② | <1 mM | 谷氨酰胺 | - | 离子去污剂(胆 酸盐、SDS) | - | 精氨酸 | - | 叠氮化钠 | 3 mM | NH₄Cl | - |
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注：
①应尽量避免使用强还原剂，防止金属离子被还原，影响蛋白结合在层析介质上。最大耐受强还原剂量根据样品不同而异，同时仅样品中含还原剂，缓冲液中不能含还原剂。
②应尽量避免使用金属离子螯合剂，防止金属离子与介质分离。最大耐受强螯合剂浓度根据样品不同而异，同时仅样品中含螯合剂，缓冲液中不能含螯合剂。
3.1 柱平衡
将层析柱连接至纯化设备，使用结合缓冲液冲洗5 CV 以上，至层析柱流出液pH、电导同结合缓冲液的pH、电导一致。平衡结束后将紫外归零。为减少金属离子脱落对层析的影响，建议在平衡前先用1 CV 含有1 M NaCl的0.5 M 咪唑清洗，然后再用纯化水清洗5 CV，最后用结合缓冲液平衡层析柱后再上样。
推荐结合缓冲液：20 mM PB, 500 mM NaCl, 20 mM 咪唑, pH 7.4, 0.22 μm 滤膜过滤。
当纯化包涵体时，结合缓冲液和样品中要添加8M尿素或6M盐酸胍。
3.2 加载样品
经0.22 / 0.45 μm 过滤后的样品溶液加载在层析柱上，上样体积取决于层析介质载量、样本溶液中与介质结合分子的浓度和其他层析条件。
3.3 柱冲洗
使用结合缓冲液冲洗5-10 CV，将该条件下不与介质结合的杂质从层析柱中洗去（至紫外检测值回归基线附近）。或根据前期实验确定的杂质淋洗条件进行柱冲洗。
3.4 洗脱
使用洗脱缓冲液冲洗层析柱，目标紫外峰升起后开始收集洗脱样品。推荐洗脱缓冲液：20 mM PB, 500 mM NaCl, 500 mM 咪唑, pH 7.4，0.22 μm 滤膜过滤。
可选择竞争性洗脱、降低pH 方式洗脱或螯合剂洗脱，根据实际情况进行选择。
竞争性洗脱：线性或逐步增加与金属离子（如Ni2+）有亲和力的物质浓度，如0-2 M NH4Cl、0-0.5 M 咪唑、0-0.5 M 组氨酸。梯度洗脱建议在结合缓冲液的恒定pH 下进行。
降低pH 洗脱：随着pH 降低，弱结合蛋白和强结合蛋白依次被洗脱。当缓冲液pH 降低至4.0 以下时，金属离子（如Ni2+）会与介质解离而达到洗脱目的。（如果目的蛋白对低pH 敏感，建议洗脱收集液中加入1/10 收集体积的1 M Tris-HCl，pH 9.0 进行中和）
螯合剂洗脱：50 mM 金属螯合剂EGTA 或EDTA 可将金属离子（如Ni2+）从介质中解离而达到洗脱目的，这种方法也可以洗脱变性或沉淀的蛋白质，该方法一般不推荐使用。
3.5 清洗和保存
用3-5 CV 含有1 M NaCl 的0.5 M 咪唑清洗，用纯化水冲洗至流出液电导为0，最后用20%乙醇冲洗2-3 CV。层析柱置于4℃环境中保存。
介质重复使用取决于样品的性质，建议每种样品使用单独的层析介质，防止交叉污染。
4. 再生
杂质残留和金属离子脱落将会影响层析性能和载量，建议1-5 个循环后进行再生，
重新螯合金属离子。再生步骤如下：
用10 CV 纯化水冲洗层析柱。
用3-5 CV 脱离子缓冲液(20 mM PB, 0.5 M NaCl, 50 mM EDTA, pH 7.4)冲洗。
用3-5 CV 结合缓冲液(不含咪唑)冲洗层析柱。
用5 CV 纯化水冲洗层析柱。
用0.5 CV 以上0.1 M 金属离子溶液（如NiSO4）按15 cm/h 流速螯合。
用3 CV 的0.5 M 乙酸钠-乙酸（pH 4.0）冲洗层析柱。
用5 CV 结合缓冲液平衡层析柱后立即使用。
用5 CV 的20%乙醇冲洗层析柱后保存。
金属离子浓度为0.1 M，溶液保持中性或偏弱酸性，0.2 μm 过滤后使用。
当螯合Ca2+时，避免使用磷酸盐缓冲液（会形成沉淀）。当脱去Fe3+时，由于Fe3+在中性溶液中容易形成不溶物，建议用50 mM EDTA过夜，以脱去Fe3+；当螯合Fe3+时，必须在较低pH（约pH 3.0）下螯合，以防止Fe3+产生沉淀。
5. 在位清洗
在位清洗（Cleaning In Place，CIP）是去除层析介质上强结合的、沉淀的或者变性的物质。杂质残留将会影响层析柱的层析性能。如果聚集严重，将会堵塞层析柱，加大反压并减慢流速。所以定期的在位清洗能够防止污染物在柱床聚集，有助于保持介质的载量，流速等。
层析柱一般在使用3-5 个循环后需要进行清洗，帮助介质恢复良好的性能。对不同类型的杂质和污染物建议清洗条件如下：
1.去除因离子交换作用吸附的蛋白：用2-3 CV 的2 M NaCl 溶液清洗层析柱，然后用结合缓冲液冲洗层析柱。
2.去除蛋白沉淀、疏水结合的物质：先脱去金属离子，然后用0.2-1 M NaOH浸泡层析柱1 h 以上。
3.去除强结合的疏水性蛋白和脂质等：用3-5 CV 的70%乙醇或者30%异丙醇冲洗层析柱（15-20 min），然后用结合缓冲液平衡层析柱。另外，6 M 盐酸胍浸泡过夜可去除大部分杂蛋白。清洗后如果立即使用，用结合缓冲液冲洗3-10 CV 即可。
6. 消毒灭菌
消毒灭菌是为了将层析柱中的微生物污染降至最低。20%乙醇不具有杀菌、除热原作用，建议用70%乙醇处理12 h 以减少微生物污染风险。或者使用0.5-1 M 的NaOH 浸泡0.5-1 h 进行消毒灭菌。
7. 保存
未使用的介质应当保存在2-8℃的干燥、通风、洁净环境中，确保容器口完全密闭，切记不可冷冻。
已装好的层析柱应当使用20%乙醇或2%苯甲醇浸泡储浸泡密封保存，防止微生物污染。为了防止乙醇挥发导致微生物滋生，建议间隔3 个月更换新鲜的保存液。
保存的介质在使用前，应进行CIP 和至少用结合缓冲液平衡5 CV。
Immobilized Metal Ion Affinity Chromatography (IMAC) separates and purifies proteins based on the specific binding between certain amino acids on the protein surface (e.g., histidine, tryptophan, cysteine) and metal ions
This product is a metal chelate affinity chromatography medium (non-metal-chelated) prepared by conjugating nitrilotriacetic acid (NTA) ligand to 6% highly cross-linked agarose. It provides 4 coordination sites for metal ion chelation, resulting in more stable chelated metal ions and tolerance to reducing agents at certain concentrations. After users chelate metal ions, it can be used for the separation and purification of proteins and peptides in biopharmaceutical and bioengineering downstream processes.
Aladdin Metal IMAC Agrose (NTA) 6FF is stored in 20% ethanol, with a gel-to-protectant volume ratio of 7:3. The product specification refers to the actual gel volume.
Parameter | Specification | Matrix | 6% Highly cross-linked agarose | Functional group | Nitrilotriacetic acid (NTA) | Average particle size | 90 μm | Metal chelation capacity | 15 μmol Ni2+/mL medium | Binding capacity | ~40 mg His-tagged protein/mL medium (after Ni2+ chelation) | Recommended flow rate | <400 cm/h | Recommended pressure | <0.3 MPa (3 bar) | Working pH | 4–8.5 | pH stability ① | 3–12 (long-term); 2–14 (short-term) | Chemical stability | Stable in common buffers; avoid chelating agents such as EDTA, citrate, etc. | Storage conditions | 20% ethanol; 2–8℃ | Shelf life | 5 years |
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Note:
① Refers to the stability of the medium without metal ion chelation. Long-term: pH range where stability is maintained without adverse effects on subsequent performance. Short-term: pH range for regeneration, cleaning-in-place and disinfection based on experience.
Instructions for Use
1. Column Packing
1.1 Preparation of Packing Buffer
Purified water, degas by ultrasonication for 15 min.
1.2 Preparation of Chromatography Medium
Calculate the required amount of chromatography medium (compression coefficient ~1.15) and weigh for standby. Transfer to packing buffer using a vacuum filtration flask. Add an appropriate amount of packing buffer to the exchanged medium to prepare a ~50% gel suspension.
1.3 Preparation of Chromatography Column
Inspect the chromatography column to ensure all components are intact and clean. Install the lower end cap, tighten the O-ring, fix the column vertically on a iron stand, and check and adjust with a level to keep the column upright. Draw packing buffer with a syringe, connect to the column outlet, slowly push out the liquid to remove air bubbles from the bottom sieve, remove the syringe and screw on the plug. Add ~2 cm height of packing buffer to the column.
1.4 Column Packing (Take a 16 mm diameter, 10 cm bed height column as an example)
Mix the gel suspension evenly, pour slowly into the column with a glass rod guide. If there is space above the liquid level, fill with packing buffer. Connect the regulator to the chromatography system, start the pump to remove air bubbles from the tubing and top sieve, then pause. Insert the regulator into the column at 45°, fix it and tighten the seal ring to avoid air bubbles. Unscrew the bottom plug, place the bottom tubing into a waste tank, set the flow rate to 60 cm/h until the medium interface is stable, then increase to 600 cm/h and gradually reduce to 300 cm/h. Maintain this flow rate for 45 min, mark the interface with a marker, and pause the system. Screw on the bottom plug, disconnect the column top from the pump, slightly loosen the seal ring on the regulator, press the end cap 3 mm below the gel surface, and tighten the seal ring. Connect the top and bottom tubing of the column to the chromatography system for column efficiency testing.
2. Column Efficiency Determination and Evaluation
Column efficiency should be tested immediately after packing. Column performance is usually evaluated by theoretical plates per meter (N/m) and peak asymmetry factor (As). Higher column efficiency indicates stronger separation ability. For accurate results, the sample volume is 1.0% of column volume, and linear flow rate is controlled at 15–30 cm/h. Load the sample close to the column inlet to minimize dilution.
Item | Acetone Method | NaCl Method | Sample | 1.0% (v/v) aqueous acetone | 0.8 M NaCl aqueous solution | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl aqueous solution | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
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Column Efficiency Calculation
Calculate N/m and As from the UV (or conductivity) curve using the following formulas:
N/m = N/L
N = 5.54(VR /Wh )2
As = b/a
Notes: L = column height; V R = 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.As should be between 0.8 and 1.8. N/m > 3000.
3.Separation and Purification
IMAC Agrose (NTA) 6FF must be chelated with metal ions before purification. Detailed chelation steps refer to 4. Regeneration.
Metal Ion Selection:Cu 2+ , Zn 2+ , Ni 2+ are commonly used. Cu 2+ and Zn 2+ are most frequently used for purifying non-tagged proteins: Cu 2+ binds strongly to some proteins, and certain proteins only bind Cu 2+ ; Zn 2+ binds weakly to some proteins and only a few proteins, often for specific protein purification. Ni 2+ is widely used for His-tagged protein purification. Co 2+ , Fe 3+ and Ca 2+ are alternatives.
Phosphate buffer is preferred at neutral to weakly alkaline pH (7.0–8.0). Avoid EDTA, citrate, etc. Tris-HCl is acceptable but not recommended when metal-protein affinity is weak. Common additives and concentrations are listed below. Low-concentration imidazole (20–40 mM) is added to binding buffer and samples to reduce non-specific adsorption of host proteins. 0.15–0.5 M NaCl must be added to eliminate ionic adsorption.
Additives That Do Not Affect Protein Binding
Additive | Common Concentration | Additive | Common Concentration | Phosphate, Tris, Borate, HEPES | 20–100 mM | Non-ionic detergents | ≤2% | NaCl | ≤2 M | Triton X-100 | ≤2% | KCl | ≤1 M | Tween-20 | ≤2% | Guanidine hydrochloride | ≤6 M | Octyl glucoside | ≤2% | Urea | ≤8 M | Dodecyl maltoside | ≤2% | Glycerol | ≤50% | C12E8, C10E6 | ≤2% | Isopropanol | ≤60% | PMSF (protease inhibitor) | 1 mM | Ethanol | ≤30% | Pepstatin | 1 μM | 1% Benzamidine (protease inhibitor) | ≤1 mM | Leupeptin | 0.5 μg/mL |
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Additives That May Disrupt Protein Binding (Avoid if Possible)
Additive | Common Concentration | Additive | Common Concentration | β-Mercaptoethanol | <20 mM | Citrate | Tolerates low concentrations | Reduced glutathione | <10 mM | Histidine | Alternative to imidazole | Strong reducing agents (DTT, TCEP, DTE) ① | <5 mM | Glycine | – | Metal chelators (EDTA, EGTA) ② | <1 mM | Glutamine | – | Ionic detergents (cholate, SDS) | – | Arginine | – | Sodium azide | 3 mM | NH₄Cl | – |
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Notes:
① Strong reducing agents are avoided to prevent metal ion reduction and impaired protein binding. Maximum tolerance varies by sample; reducing agents only in samples, not in buffers.
② Metal chelators are avoided to prevent metal ion detachment from the medium. Maximum tolerance varies by sample; chelators only in samples, not in buffers.
3.1 Column Equilibration
Connect the column to the purification system, wash with ≥5 column volumes (CV) of binding buffer until the effluent pH and conductivity match the binding buffer. Zero the UV detector after equilibration. To reduce metal ion leaching, pre-wash with 1 CV of 0.5 M imidazole containing 1 M NaCl, then 5 CV of pure water, and equilibrate with binding buffer before loading.
Recommended binding buffer: 20 mM PB, 500 mM NaCl, 20 mM imidazole, pH 7.4, filtered through 0.2 μm membrane. For inclusion body purification, add 8 M urea or 6 M guanidine hydrochloride to binding buffer and samples.
3.2 Sample Loading
Load the sample solution filtered through 0.22/0.45 μm membrane onto the column. Loading volume depends on medium capacity, target molecule concentration, and other chromatographic conditions.
3.3 Column Washing
Wash with 5–10 CV of binding buffer to remove unbound impurities (until UV returns to baseline). Alternatively, follow impurity washing conditions determined in preliminary experiments.
3.4 Elution
Start collecting eluted fractions when the target UV peak rises.Recommended elution buffer: 20 mM PB, 500 mM NaCl, 500 mM imidazole, pH 7.4, filtered through 0.2 μm membrane.Elution methods: competitive elution, pH reduction, or chelator elution.
Competitive elution: Linearly or stepwise increase concentrations of metal-binding agents (0–2 M NH4Cl, 0–0.5 M imidazole, 0–0.5 M histidine). Gradient elution at constant pH of binding buffer is recommended.
pH reduction elution: Weakly and strongly bound proteins elute sequentially as pH drops. Metal ions (e.g., Ni2+) dissociate below pH 4.0. (If target protein is pH-sensitive, add 1/10 volume of 1 M Tris-HCl, pH 9.0 to eluate for neutralization.)
Chelator elution: 50 mM EGTA or EDTA dissociates metal ions from the medium, eluting denatured/precipitated proteins. Not generally recommended.
3.5 Cleaning and Storage
Wash with 3–5 CV of 0.5 M imidazole containing 1 M NaCl, rinse with pure water until effluent conductivity is 0, then wash with 2–3 CV of 20% ethanol. Store the column at 4℃.Reuse depends on sample properties; use dedicated medium per sample to avoid cross-contamination.
4. Regeneration
Impurity accumulation and metal ion leaching reduce performance. Regenerate and re-chelate metal ions after 1–5 cycles.
Steps:
Wash the column with 10 CV of pure water.
Wash with 3–5 CV of deionization buffer (20 mM PB, 0.5 M NaCl, 50 mM EDTA, pH 7.4).
Wash with 3–5 CV of binding buffer (without imidazole).
Wash with 5 CV of pure water.
Chelate with ≥0.5 CV of 0.1 M metal ion solution (e.g., NiSO4 ) at 15 cm/h.
Wash with 3 CV of 0.5 M sodium acetate-acetic acid (pH 4.0).
Equilibrate with 5 CV of binding buffer and use immediately.
Wash with 5 CV of 20% ethanol for storage.
Metal ion solution: 0.1 M, neutral to weakly acidic pH, filtered through 0.22 μm membrane.
Avoid phosphate buffer when chelating Ca 2+ (precipitation risk).
For Fe 3+ removal: incubate overnight with 50 mM EDTA (Fe 3+ precipitates at neutral pH).
For Fe 3+ chelation: chelate at low pH (~pH 3.0) to prevent precipitation.
5. Cleaning-In-Place (CIP)
CIP removes strongly bound, precipitated or denatured substances. Accumulated impurities impair performance, increase backpressure and reduce flow rate. Clean after 3–5 cycles to restore capacity and flow.
Ion-exchange adsorbed proteins: Wash with 2–3 CV of 2 M NaCl, then re-equilibrate with binding buffer.
Protein precipitates/hydrophobic substances: Remove metal ions first, then soak with 0.2–1 M NaOH for ≥1 h.
Strongly bound hydrophobic proteins/lipids: Wash with 3–5 CV of 70% ethanol or 30% isopropanol (15–20 min), then re-equilibrate. Soak overnight with 6 M guanidine hydrochloride to remove most impurities. Re-equilibrate with 3–10 CV of binding buffer if used immediately.
6. Disinfection and Sterilization
To minimize microbial contamination:
Treat with 70% ethanol for 12 h (20% ethanol has no bactericidal/pyrogen-reducing effect).
Or soak with 0.5–1 M NaOH for 0.5–1 h.
7. Storage
Unused medium should be stored in a dry, well-ventilated and clean environment at 2–8°C. Ensure the container is tightly sealed; do not freeze under any circumstances. Packed chromatography columns shall be immersed and hermetically stored in 20% ethanol or 2% benzyl alcohol to prevent microbial contamination. To avoid microbial growth caused by ethanol volatilization, it is recommended to replace the storage solution with fresh one every 3 months. Prior to use, the stored medium shall undergo CIP treatment and be equilibrated with binding buffer for at least 5 column volumes (CV).
ZJ26F0838611
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