计算溶液所需的质量、体积或浓度。
苯甲脒亲和层析介质
有货
为什么选择此级别
BioReagent,通过内毒素测试 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,禁止冷冻。低温运输,禁止冷冻 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
本产品是一种亲和层析介质，基质为4%高度交联琼脂糖，具有可靠的物理化学稳定性，可使用较高流速。苯甲脒亲和层析介质的配基为对氨基苯甲脒 (p-aminobenzamidine)，通过带酰胺键的间臂共价结合到琼脂糖上。苯甲脒类物质是丝氨酸蛋⽩酶的⼴谱抑制剂，因此本产品常⽤于丝氨酸蛋⽩酶、胰蛋⽩酶、尿激酶、凝⾎酶、激肽释放酶等的分离纯化或从⽣物样品中去除这些物质。
阿拉丁苯甲脒亲和层析介质储存在含50mM 乙酸钠-乙酸（pH4）的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 | 4%高度交联琼脂糖 |
| 配基 | 对氨基苯甲脒，＞12 μmol/mL 介质 |
| 平均粒径 | 90 μm |
| 结合载量 | ＞35mg 胰蛋白酶/mL 介质 |
| 建议压力 | ＜0.15 MPa（1.5 bar） |
| 建议流速 | ＜250 cm/h |
| pH 稳定性 | 2-8（长期①）；1-9（短期②） |
| 化学稳定性 | 在常用水溶性缓冲液中稳定 |
| 储存 | 含50mM 乙酸钠-乙酸（pH4）的20%乙醇；2-8℃ |
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。
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75%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2cm高的装柱缓冲液。
1.4 装柱（以直径16 mm，15 cm柱床高度为例）
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60cm/h，至介质界面无变化后，设置流速为400cm/h，维持该流速45min，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。
2. 柱效检测
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。
| 方法 | 丙酮检测 | NaCl检测 |
| 平衡液 | 纯化水 | 0.4 M NaCl |
| 样品 | 1%丙酮水溶液，1.0% CV | 0.8 M NaCl，1.0% CV |
| 流速 | 30 cm/h | 30 cm/h |
| 检测信号 | UV-280 nm | 电导 |
注：CV (Column Volume) 柱体积，以下简称CV。
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 (Vʀ / Wₕ)²
As = b / a
备注：
- Vʀ：保留体积；
- Wₕ：半高峰宽；
- L：柱高(单位为m)；
- N：理论塔板数。
Vʀ和Wₕ的单位应一致；a是10%峰高处的第一个半峰宽；b是10%峰高处的第二个半峰宽。
峰应该是对称的，As尽可能接近1（通常可接受0.8-1.8之间的值）。N/m＞3,000。
3、分离纯化
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。
3.2 加载样品
经0.2 / 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。
3.3 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。
3.5 清洗和保存
用1.0% Triton X-100 冲洗5 CV，用纯化水冲洗10 CV。然后用6 M盐酸胍冲洗 5 CV，用纯化水冲洗至流出液电导为0。最后使用含50mM 乙酸钠-乙酸（pH4） 的20% 乙醇冲洗5 CV，置于2-8℃保存。
备注：
- 推荐的结合缓冲液：50mM Tris-HCl, 0.5M NaCl, pH7.4。结合缓冲液经0.2 μm过滤。
- 推荐的洗脱缓冲液：50mM 甘氨酸, pH3.0；或10mM HCl, 0.5M NaCl, pH2.0；或50 mM Tris-HCl, 0.5 M NaCl, 20 mM p-aminobenzamidine, pH7.4。洗脱缓冲液经0.2 μm过滤。
- 若采用低pH洗脱缓冲液，收集洗脱液后立即按1mL洗脱液加入60-200μL 1M Tris-HCl，pH9.0，防止洗脱液中的蛋白在低pH下变性。
- 若采用竞争性洗脱缓冲液，即在结合缓冲液中加入 20 mM p-aminobenzamidine，收集的洗脱液中不需加入Tris-HCl，pH9.0调pH。
4、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。层析柱在每次使用后均应进行清洗。短期内介质可在pH1-9的范围内使用，介质在pH9溶液中保存一周（168h），其载量仍保持在一半以上。然而，介质持续 暴露于高于pH8或低于pH2的缓冲液中，会引起配基的水解或分解。推荐使用盐酸胍去除结合于介质的沉淀或变性物质。对于结合于介质上的疏水性物质，可使用非离子表面活性剂或乙醇去除。
5、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。苯甲脒亲和层析介质的消毒灭菌可以用含有2%氯己定和20%乙醇的缓冲液平衡，并维持6h，然后用无菌结合缓冲液清洗至少5 CV；或用70%乙醇平衡，并维持12h，然后用无菌结合缓冲液清洗至少5 CV。
6、保存
- 未使用的介质应当保存在2-8℃干燥、通风、洁净环境中，确保容器口完全密闭。
- 已使用的介质或层析柱应保存在含50mM 乙酸钠-乙酸（pH4）的20%乙醇中，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。
7、线性放大
在实验室规模进行优化后的纯化工艺，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：
7.1 保持停留时间不变，保证动态载量的稳定。
7.2 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。
7.3 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 10-25 cm。
7.4 保证样品浓度均匀一致，洗脱条件一致。
常见问题及解决方法
1、使用过程中压力过高
- 更换在线滤器
- 层析柱堵塞，进行CIP清理
2、压力曲线不稳
- 排除上样泵里的气泡
- 使用气泡陷阱去除样品中的气泡
3、洗脱峰逐渐变宽
- 层析柱可能被污染，优化洗脱条件，并严格按CIP进行清洗
4、回收率降低
- 上样量过高，适当减少上样量
- 洗脱时发生沉淀，优化洗脱条件
- 洗脱强度不够，优化洗脱条件
5、CIP峰增大
- CIP 强度不够，优化CIP条件
6、首次使用配基脱落较高
- 首次使用时进行一次空载运行，并进行CIP
This product is an affinity chromatography medium with a matrix of 4% highly cross-linked agarose, offering reliable physicochemical stability and suitability for high flow rates. The ligand of the benzamidine affinity medium is p-aminobenzamidine, covalently coupled to agarose via an amide-bond spacer arm. Benzamidine compounds are broad-spectrum inhibitors of serine proteases. Therefore, this product is commonly used for the separation and purification of serine proteases, trypsin, urokinase, thrombin, kallikrein, etc., or for the removal of these substances from biological samples.
Aladdin Benzamidine Agarose Resin is stored in 20% ethanol containing 50 mM sodium acetate-acetic acid (pH 4.0), with a settled gel to storage solution ratio of 7:3. The product specification refers to the actual volume of the settled gel.
| Parameter | Specification |
| Matrix | 4% Highly Cross-linked Agarose |
| Ligand | p-Aminobenzamidine, >12 μmol/mL medium |
| Average Particle Size | 90 μm |
| Binding Capacity | >35 mg Trypsin / mL medium |
| Recommended Pressure | <0.15 MPa (1.5 bar) |
| Recommended Flow Rate | ＜250 cm/h |
| pH Stability¹ | 2-8 (long term); 1-9 (short term) |
| Chemical Stability | Stable in common aqueous buffers |
| Storage | In 20% ethanol containing 50 mM sodium acetate-acetic acid (pH 4.0); 2-8°C |
Note1:
Long term: pH range where the medium remains stable for extended periods without adversely affecting its subsequent performance.
Short term: pH range empirically recommended for regeneration, cleaning-in-place (CIP), and sanitization.
Instructions for Use
1. Column Packing
1.1 Packing Buffer Preparation
Prepare purified water. Degas by sonication for 15 min.
1.2 Chromatography Medium Preparation
Calculate the required amount of medium (compression factor ~1.15) and weigh it out. Transfer it to the packing buffer using a vacuum filtration flask. Add packing buffer to the exchanged medium to create a ~75% slurry.
1.3 Chromatography Column Preparation
Inspect the column to ensure all parts are intact and clean. Attach the bottom adaptor, tighten the O-ring, and secure the column vertically on a stand. Check and adjust for vertical alignment using a level. Use a syringe to draw packing buffer, connect it to the bottom outlet, and slowly push the liquid to remove air bubbles from the bottom screen. Remove the syringe and attach the end cap. Add packing buffer to the column to a height of ~2 cm.
1.4 Packing (Example for a column with diameter 16 mm, bed height 15 cm)
Mix the slurry thoroughly. Slowly pour it into the column using a glass rod as a guide. If there is space left at the top, fill it with packing buffer. Connect the adaptor to the chromatography system. Start the pump to purge air bubbles from the tubing and top screen at a certain flow rate, then pause. Insert the adaptor into the column at a 45° angle, secure it, and tighten the seal, avoiding air bubbles.
Unscrew the bottom end cap and place the outlet tubing into a waste container. Set the flow rate to 60 cm/h until the bed interface stabilizes. Then set the flow rate to 400 cm/h and maintain for 45 min. Mark the interface with a pen and pause the system. Attach the bottom end cap. Disconnect the top of the column from the pump. Slightly loosen the adaptor seal, lower the adaptor to 2 mm below the bed surface, and tighten the seal. Connect the top and bottom tubing of the column to the chromatography system for column efficiency testing.
2. Column Efficiency Test
Column efficiency should be tested immediately after packing. Terms such as Number of Theoretical Plates per meter (N/m) and Peak Asymmetry Factor (As) are commonly used to evaluate column performance. Higher column efficiency indicates better separation capability. For accurate assessment, the sample volume should be 1.0% of the column volume (CV), and the linear flow rate should be controlled between 15-30 cm/h. To prevent sample dilution, inject the sample as close to the column inlet as possible.
| Method | Acetone Test | NaCl Test |
| Equilibration Buffer | Purified Water | 0.4 M NaCl |
| Sample | 1% Acetone in H₂O, 1.0% CV | 0.8 M NaCl, 1.0% CV |
| Flow Rate | 30 cm/h | 30 cm/h |
| Detection Signal | UV 280 nm | Conductivity |
Note: CV (Column Volume)
Column Efficiency Calculation
Calculate N/m and As from the UV curve (or conductivity curve) using the following formulas:
N/m = N / L
N = 5.54 (Vʀ / Wₕ)²
As = b / a
Where:
Vʀ: Retention Volume
Wₕ: Peak Width at Half Height
L: Column Height (in meters)
N: Number of Theoretical Plates
Vʀ and Wₕ must have the same units.
a: Width of the first half of the peak at 10% peak height
b: Width of the second half of the peak at 10% peak height
The peak should be symmetrical, with As as close to 1 as possible (values between 0.8 and 1.8 are generally acceptable). N/m should be > 3,000.
3. Separation and Purification
3.1 Column Equilibration
Connect the column to the purification system. Equilibrate with Binding Buffer for more than 3-5 CV until the pH and conductivity of the column effluent match those of the Binding Buffer. Zero the UV detector after equilibration.
3.2 Sample Loading
Load the sample solution (filtered through 0.2 / 0.45 μm) onto the affinity column. The loading volume depends on the medium's binding capacity, the concentration of the molecule binding to the medium in the sample solution, and other chromatographic conditions.
3.3 Column Washing
Wash the column with 3-5 CV of Binding Buffer to remove impurities that do not bind under these conditions (until the UV signal returns to baseline). Alternatively, use washing conditions determined from prior experiments.
3.4 Elution
Elute the column using Elution Buffer. Start collecting fractions when the target UV peak begins to rise.
3.5 Cleaning and Storage
Wash the column with 5 CV of 1.0% Triton X-100, followed by 10 CV of purified water. Then wash with 5 CV of 6 M Guanidine Hydrochloride, and rinse with purified water until the effluent conductivity is 0. Finally, rinse with 5 CV of 20% ethanol containing 50 mM sodium acetate-acetic acid (pH 4.0) and store at 2-8°C.
Notes:
*Recommended Binding Buffer: 50 mM Tris-HCl, 0.5 M NaCl, pH 7.4. Filter through 0.2 μm.*
*Recommended Elution Buffers: 50 mM Glycine, pH 3.0; or 10 mM HCl, 0.5 M NaCl, pH 2.0; or 50 mM Tris-HCl, 0.5 M NaCl, 20 mM p-aminobenzamidine, pH 7.4. Filter through 0.2 μm.*
*If using a low pH elution buffer, immediately add 60-200 μL of 1 M Tris-HCl, pH 9.0 per 1 mL of eluate after collection to prevent protein denaturation at low pH.*
*If using competitive elution (buffer with 20 mM p-aminobenzamidine), adding Tris-HCl, pH 9.0 to the eluate is not necessary.*
4. Cleaning In Place (CIP)
CIP removes strongly bound, precipitated, or denatured substances from the chromatography medium. Residual impurities can affect column performance. Severe buildup can clog the column, increasing backpressure and reducing flow rate. Regular CIP prevents contaminant accumulation and helps maintain the medium's binding capacity and flow properties. The column should be cleaned after each use. The medium can be used within pH 1-9 for short periods. The medium retains over half its binding capacity after storage in pH 9 solution for one week (168h). However, continuous exposure to buffers above pH 8 or below pH 2 may cause ligand hydrolysis or decomposition. Guanidine hydrochloride is recommended for removing precipitated or denatured substances bound to the medium. Hydrophobic substances bound to the medium can be removed using non-ionic surfactants or ethanol.
5. Sanitization
Sanitization minimizes microbial contamination in the column. The benzamidine affinity medium can be sanitized by equilibrating with a buffer containing 2% chlorhexidine and 20% ethanol for 6 hours, followed by washing with at least 5 CV of sterile Binding Buffer; or by equilibrating with 70% ethanol for 12 hours, followed by washing with at least 5 CV of sterile Binding Buffer.
6. Storage
Unused medium should be stored in a dry, ventilated, clean environment at 2-8°C, ensuring the container is tightly sealed.
Used medium or packed columns should be stored submerged in 20% ethanol containing 50 mM sodium acetate-acetic acid (pH 4.0) to prevent microbial growth.
To prevent microbial growth due to ethanol evaporation, replace the storage solution with fresh solution every 3 months.
Before using stored medium, perform CIP and equilibrate with at least 5 CV of Binding Buffer.
7. Linear Scale-Up
Purification processes optimized at laboratory scale can be linearly scaled up to pilot or production scale. Consider the following during scale-up:
7.1 Maintain the same residence time to ensure consistent dynamic binding capacity.
7.2 Select the column volume based on the required binding capacity. Note if changes in bed height affect the purification steps.
7.3 Determine the column diameter based on flow rate requirements. Determine the bed height based on the known residence time; 10-25 cm is generally recommended.
7.4 Ensure sample concentration homogeneity and consistent elution conditions.
Troubleshooting
规格
别名
苯甲脒琼脂糖凝胶4FF
英文别名
Benzamidine beads
规格或纯度
BioReagent, 通过内毒素测试, 70% v/v
稳定性与储存
Store at 2-8℃ long term (60 months). Do not freeze.
英文名称
Benzamidine Agarose Resin
储存条件
2-8°C储存,禁止冷冻
运输条件
低温运输,禁止冷冻
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1029052 |
分析证书 | B1501883 |
| ZJ25F1029051 |
分析证书 | B1501883 |
技术文档和文章
溶液计算器
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