储存缓冲液：100% (v/v) Acetone
溴化氰活化亲和层析介质 4FF是一种 CNBr 预活化的琼脂糖介质，基质为 4% 高度交联琼脂糖，具备良好的物理和化学稳定性；介质偶联简单直接、反应迅速，条件温和，可直接偶联含伯氨基的蛋白及其他生物大分子，无需偶联间隔臂，还能形成多点偶联、配基不易脱落；同时亲水性强、非特异吸附少，通用性与适用范围广，且流速快、重复性好、易于工艺放大，主要应用于蛋白等生物分子的分离纯化。
阿拉丁溴化氰活化亲和层析介质 4FF储存在100%丙酮中，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 | 4%高度交联琼脂糖 | 偶联官能团 | -NH₂ | 活化基团 | CNBr | 平均粒径 | 90 μm | 偶联条件 | pH 8-10，室温（20-25℃）2-3 h或4℃过夜 | 偶联载量 | 13-26 mg α-胰凝乳蛋白酶原/mL介质 | 建议流速 | <250 cm/h | 建议压力 | <0.15 MPa（1.5 bar） | pH稳定性 ① | 3-11（长期）；2-11（短期） | 化学稳定性 | 偶联配基后在常用水溶性缓冲液中稳定，可使用8 M 尿素或6 M 盐酸胍等变性剂② | 保存条件 | 丙酮；-15℃以下 | 保质期 | 1年 |
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注：
① 长期，指介质在很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，指根据经验给出的介质再生、在位清洗和消毒的pH 范围。
② 偶联配基后的稳定性是在配基能够承受pH 值或化学环境变化的前提下。
使用说明
1.偶联功能配基
缓冲液 | 体积 | Solution A：1 mM HCl, 0.5 M NaCl（需预冷，pH2-3） | ≥20 倍胶体积 | Solution B：0.2 M NaHCO₃, 0.5 M NaCl, pH8.3 | ≥10 倍胶体积 | Solution C：0.1 M Tris-HCl, pH8.0 | ≥5 倍胶体积 | Solution D：0.05 M Tris-HCl, 0.5 M NaCl, pH8-9 | ≥15 倍胶体积 | Solution E：0.1 M 乙酸-乙酸钠, 0.5 M NaCl, pH3-4 | ≥15 倍胶体积 | Solution F：0.02 M PB, 0.15 M NaCl, pH7.0-7.6 | ≥10 倍胶体积 |
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1.2 配基预处理：通常1 mL 介质可偶联约5-10 mg 蛋白。干粉状蛋白配基可用Solution B 溶解，溶液状态的蛋白配基则需将其缓冲液置换为Solution B。
1.3 取适量丙酮中保存的亲和层析介质放于玻璃过滤漏斗中，用15 倍胶体积预冷Solution A 洗涤。
1.4 洗涤完成后，将介质加至配基溶液中混匀，室温反应2-4 h 或4℃反应过夜。
1.5 偶联反应完成后，使用5 倍胶体积Solution B 洗涤，去除多余未偶联配基。
1.6 使用3 倍胶体积Solution C 洗涤后，继续用Solution C 溶液封闭介质剩余的活化位点，室温封闭2-4 h。
1.7 Solution D 与Solution E 交替洗涤3-6 次，每次用3 倍胶体积洗涤。
1.8 最后使用10 倍胶体积Solution F 洗涤，完成功能配基的偶联。
偶联注意事项
偶联缓冲液：碳酸氢盐或硼酸盐缓冲液是常用的偶联溶液。勿使用Tris 或其他含氨基的缓冲液作为偶联缓冲液；为保持较高的偶联效率，pH 通常选择8-10；低pH 会影响介质偶联活性，但有时为减小空间位阻效应或保留配基生物学活性，偶联反应需在低pH 下进行，如pH 6。
为减小蛋白之间的相互吸附，可在偶联溶液中加入适当浓度的盐，如0.5 M NaCl。温度与反应时间：室温条件下（如25℃），交联反应2-3 h 内即可完成。若偶联需要在低温条件下（如4℃）进行，可过夜反应（或反应10-16 h）。
配基浓度：建议1 mL 介质加入5-10 mg 蛋白配基。配基添加量过多可能会导致介质配基密度过高，而出现空间位阻效应，降低介质吸附量。同时，配基密度过高还会增加洗脱难度和介质的非特异性吸附。
2.装柱
2.1 装柱缓冲液准备
纯化水，超声脱气15 min。
2.2 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75%浓度的胶悬液。
2.3 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。
2.4 装柱（以直径16 mm，床高15 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400 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2 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。
3.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
4.分离纯化
4.1 柱平衡
将层析柱连接至纯化设备，使用结合缓冲液冲洗3-5 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。
4.2 加载样品
经0.22/ 0.45 μm 过滤后的样品溶液加载在亲和层析柱上，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。
4.3 柱冲洗
使用结合缓冲液冲洗3-5 CV，将该条件下不与介质结合的杂质洗去（至UV 280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。
4.4 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。
4.5 清洗和保存
常用清洗再生方法：高pH 缓冲液（如0.1 M Tris-HCl, 0.5 M NaCl, pH8.5）与低pH 缓冲液（如0.1 M 乙酸钠-乙酸, 0.5 M NaCl, pH4.5）交替洗涤3-5 次，
每次用2-3 CV 洗涤。最后用结合缓冲液平衡3-5 CV。然后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2-8℃环境中保存。清洗再生方法因偶联配基而异，此方法仅供参考。缓冲液种类、结合、洗脱条件，与偶联配体密切相关；缓冲液需经0.22 μm 过滤。样本的缓冲液应尽量与平衡缓冲液相同或相近。
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。在位清洗的频率取决于起始样品的性质和条件，建议1-5 个循环需要进行一次在位清洗。
如果配基稳定性允许，偶联后介质可长期暴露在0.1 M NaOH 中。对于不同类型的杂质和污染物建议清洗条件如下：
沉淀或变性物质：0.1 M NaOH 清洗4 CV（接触时间1-2 h），结合缓冲液清洗至少5 CV；或6 M 盐胍酸清洗2 CV，然后立即用结合缓冲液清洗至少5 CV。
疏水型杂质：0.1-0.5%非离子去污剂清洗2 CV，然后立即用结合缓冲液清洗至少5 CV；或70%乙醇清洗3-4 CV，然后立即用结合缓冲液清洗至少5 CV。
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。可以用70%乙醇平衡（配基耐受该条件的情况下），并维持12 h，然后用无菌结合缓冲液清洗至少5 CV。
7.保存
未使用的介质应当保存在-15℃以下避光、干燥、通风、洁净环境中，确保容器口完全密闭。短期保存可避光置于4℃。
已偶联配基的介质或层析柱应用20%乙醇或者2%苯甲醇浸泡密封保存，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。
8.线性放大
在实验室规模优化后的纯化工艺，可以线性放大到中试或生产规模。在放大过程中需要注意以下事项：
保持停留时间不变，保证动态载量的稳定。根据需要的结合能力选择层析柱容积，如果柱高改变需要注意是否对纯化步骤产生影响。根据流速需求确定层析柱直径，根据已知的停留时间确定柱床高度，一般推荐3-15 cm。
保证样品浓度均匀一致，洗脱条件一致。
CNBr-activated Agrose 4FF is a pre-activated agarose medium with 4% highly cross-linked agarose as the matrix, featuring excellent physical and chemical stability. The medium enables simple, direct, rapid and mild coupling, and can directly conjugate proteins and other biological macromolecules containing primary amino groups without a coupling spacer arm. It also allows multi-point coupling, making ligands not easy to detach. Meanwhile, it has strong hydrophilicity, low non-specific adsorption, wide universality and application scope, fast flow rate, good repeatability and easy process scale-up. It is mainly used for the separation and purification of biological molecules such as proteins.
Aladdin CNBr-activated Agrose 4FF is stored in acetone, with a volume ratio of gel to protective solution of 7:3. The product specification is based on the actual gel volume.
Parameter Specifications
Item | Specification | Matrix | 4% Highly cross-linked agarose | Coupling functional group | -NH₂ | Activated group | CNBr | Average particle size | 90 μm | Coupling conditions | pH 8–10, room temperature (20–25℃) for 2–3 h or 4℃ overnight | Coupling capacity | 13–26 mg α-chymotrypsinogen/mL medium | Recommended flow rate | <250 cm/h | Recommended pressure | <0.15 MPa (1.5 bar) | pH stability ① | 3–11 (long-term); 2–11 (short-term ) | Chemical stability | Stable in common aqueous buffers after ligand coupling; denaturants such as 8 M urea or 6 M guanidine hydrochloride can be used ② | Storage conditions | Acetone; below -15℃ | Shelf life | 1 year |
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Notes:
① Long-term refers to the pH range where the medium remains stable for a long time without adverse effects on its subsequent performance. Short-term refers to the pH range for medium regeneration, cleaning-in-place and disinfection based on experience.
② The stability after ligand coupling is premised on the ligand’s tolerance to changes in pH or chemical environment.
Instructions for Use
1. Coupling of Functional Ligands
1.1 Buffer Volume
Solution | Composition | Volume Ratio | Solution A | 1 mM HCl, 0.5 M NaCl (pre-cooled, pH 2–3) | ≥20× gel volume | Solution B | 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3 | ≥10× gel volume | Solution C | 0.1 M Tris-HCl, pH 8.0 | ≥5× gel volume | Solution D | 0.05 M Tris-HCl, 0.5 M NaCl, pH 8–9 | ≥15× gel volume | Solution E | 0.1 M Acetic acid-sodium acetate, 0.5 M NaCl, pH 3–4 | ≥15× gel volume | Solution F | 0.02 M PB, 0.15 M NaCl, pH 7.0–7.6 | ≥10× gel volume |
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1.2 Dissolve dry powder protein ligand with Solution B; replace the buffer of solution-state protein ligand with Solution B.
1.3 Place an appropriate amount of affinity chromatography medium stored in acetone in a glass filter funnel, and wash with 15× gel volume of pre-cooled Solution A.
1.4 After washing, add the medium to the ligand solution, mix well, and react at room temperature for 2–4 h or at 4℃ overnight.
1.5 After coupling, wash with 5× gel volume of Solution B to remove excess uncoupled ligand.
1.6 Wash with 3× gel volume of Solution C, then continue to block remaining active sites of the medium with Solution C at room temperature for 2–4 h.
1.7 Wash alternately with Solution D and Solution E for 3–6 cycles, 3× gel volume each time.
1.8 Finally, wash with 10× gel volume of Solution F to complete ligand coupling.
Coupling Notes
Coupling buffer: Bicarbonate or borate buffer is commonly used. Do not use Tris or other amino-containing buffers as coupling buffer; pH 8–10 is recommended for high coupling efficiency. Low pH affects coupling activity, but pH 6 may be used to reduce steric hindrance or preserve ligand bioactivity.
Add 0.5 M NaCl to reduce protein-protein adsorption.
Temperature & time: 2–3 h at room temperature (25℃); overnight (10–16 h) at 4℃.
Ligand concentration: 5–10 mg protein per mL medium is recommended. Excess ligand causes high ligand density, steric hindrance, reduced binding capacity, difficult elution and increased non-specific adsorption.
2.Column Packing
2.1 Packing Buffer Preparation
Purified water, ultrasonic degassing for 15 min.
2.2 Chromatography Medium Preparation
Calculate required medium volume (compression factor ~1.15) and weigh. Replace buffer to packing buffer via vacuum filtration. Prepare ~75% gel suspension with packing buffer.
2.3 Chromatography Column Preparation
Inspect column for integrity and cleanliness. Install lower end cap, tighten O-ring, fix column vertically and verify with level gauge. Remove air bubbles from bottom sieve with packing buffer via syringe, then cap. Add ~2 cm height of packing buffer to column.
2.4 Column Packing (Example: 16 mm diameter, 15 cm bed height)
Mix gel suspension, pour slowly into column with glass rod; fill with packing buffer if needed. Connect regulator to system, purge air bubbles at set flow rate then pause. Insert regulator at 45° and seal. Open bottom cap, set flow rate to 60 cm/h until bed interface stabilizes, then increase to 400 cm/h for 45 min, mark interface. Pause system, cap bottom, disconnect top, loosen regulator seal, lower upper end cap to 2 mm below gel surface, retighten. Connect column to system for column efficiency test.
3.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; 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.As should be between 0.8 and 1.8. N/m > 3000.
4. Separation and Purification
4.1 Column Equilibration
Flush column with binding buffer for ≥3–5 CV until effluent pH and conductivity match binding buffer; zero UV detector.
4.2 Sample Loading
Load sample filtered through 0.2/0.45 μm membrane. Loading volume depends on medium capacity, target molecule concentration and conditions.
4.3 Column Washing
Wash with binding buffer for 3–5 CV until UV 280 returns to baseline (or use optimized washing conditions).
4.4 Elution
Elute with elution buffer; collect fractions when target UV peak appears.
4.5 Cleaning and Storage
Regeneration: Alternate high-pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low-pH (0.1 M sodium acetate-acetic acid, 0.5 M NaCl, pH 4.5) buffers for 3–5 cycles (2–3 CV each). Equilibrate with binding buffer (3–5 CV), flush with pure water to zero conductivity, then flush with 20% ethanol (2–3 CV). Store at 2–8℃.
Conditions vary by ligand; all buffers must be 0.22 μm filtered. Sample buffer should match equilibration buffer.
5. Cleaning-In-Place (CIP)
CIP removes strongly bound, precipitated or denatured contaminants that impair performance, increase backpressure and reduce flow rate. Recommended every 1–5 cycles.
Precipitated/denatured contaminants: 0.1 M NaOH (4 CV, 1–2 h contact) → binding buffer (≥5 CV); or 6 M guanidine hydrochloride (2 CV) → immediate binding buffer wash (≥5 CV).
Hydrophobic impurities: 0.1–0.5% non-ionic detergent (2 CV) → binding buffer (≥5 CV); or 70% ethanol (3–4 CV) → immediate binding buffer wash (≥5 CV).
6. Sterilization
Minimize microbial contamination with 70% ethanol (if ligand-tolerant) for 12 h, then flush with sterile binding buffer (≥5 CV).
7. Storage
Unused medium: Store below -15℃, protected from light, dry, ventilated and sealed; short-term storage at 4℃ (light-proof).
Coupled medium/column: Store sealed in 20% ethanol or 2% benzyl alcohol. Replace storage solution every 3 months to prevent microbial growth.
8. Linear Scale-Up
Lab-scale purification can be linearly scaled to pilot or production scale with these guidelines:
Keep residence time constant for stable dynamic binding capacity.
Select column volume based on binding demand; adjust bed height if needed.
Determine column diameter by flow rate; recommended bed height: 3–15 cm.
Maintain uniform sample concentration and consistent elution conditions.
ZJ26F0636351
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