计算溶液所需的质量、体积或浓度。
超螺旋质粒DNA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
亲和层析(Affinity Chromatography，AC)是利用生物分子间专一的亲和力而进行分离的一种层析技术，当样品流经固定相时，待分离的生物分子与配基发生特异性的结合而留在固定相上，杂质不能与配基结合，随洗脱液流出，实现生物分子的分离纯化。亲和层析是分离纯化蛋白质、酶等生物大分子最常用的层析技术之一，分离过程简单、快速，也常用于生物大分子结构和功能研究。
本产品是将 2-巯基吡啶偶联⾄高刚性琼脂糖基质上制备⽽成的⼀种嗜硫亲和层析介质。嗜硫亲和层析的原理是利用电子供体和电子受体之间的相互作用来分离纯化生物分子，这种作用在高盐环境下得以加强，在低盐环境下减弱。本产品主要用于超螺旋质粒DNA的纯化。纯化过程中， 超螺旋质粒DNA吸附于介质上，开环质粒DNA不与介质结合，从而达到分离的效果。纯化后的高质量超螺旋质粒DNA常用于基因治疗或用作DNA疫苗。
阿拉丁超螺旋质粒DNA亲和层析介质储存在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 | 高刚性琼脂糖 |
| 配基 | 2-巯基吡啶 |
| 平均粒径 | 40 μm |
| 结合载量 | ~3mg 超螺旋质粒DNA/mL介质 |
| 建议流速 | ＜300 cm/h |
| 建议压力 | ＜0.5 MPa (5 bar) |
| pH 稳定性① | 3-11（长期）；2-13（短期） |
| 化学稳定性 | 常用水溶性缓冲液中稳定，如70%乙醇、0.1 M NaOH、1 M 乙酸等 |
| 储存 | 20%乙醇，4-30℃ |
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2cm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60cm/h，至介质界面无变化后，设置流速为1200cm/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1-3 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＞9,000。
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2-3 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。
备注：结合缓冲液：100 mM Tris-HCl, 2.1 M (NH₄)₂SO₄, 10 mM EDTA, pH7.5，0.2 μm过滤。
3.2 加载样品
加载经0.45 μm滤膜过滤后的样品（含超螺旋质粒DNA）到层析柱上，上样体积取决于层析介质载量、样本溶液中超螺旋质粒DNA浓度和其他层析条件。去 RNA 后的样品可直接上样。其他样品需加盐和酸（或碱）来调节电导、pH使其与结合缓冲液尽量保持一致，经0.45 μm滤膜过滤后上样。
备注：建议加载样品流速＜150 cm/h，柱高越大流速越慢。
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。
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。
备注：洗脱缓冲液：100mM Tris-HCl, 1.7 M (NH₄)₂SO₄, 10 mM EDTA, 0.3 M NaCl, pH7.5，0.2 μm过滤。
3.5 清洗和保存
用3 CV纯化水清洗，再用3 CV 0.5 M NaOH清洗3-5 CV，然后纯化水冲洗层析柱至中性，最后用20%乙醇冲洗2-3 CV。层析柱置于4-30℃环境中保存。
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。常用在位清洗方法如下：
4.1 去除变性蛋白：层析柱使用0.5 M NaOH清洗3-5 CV，然后立即用至少5 CV的结合缓冲液冲洗。
4.2 去除强疏水性蛋白或脂类：层析柱使用20 mM PB，30%异丙醇，pH7.5清洗缓冲液冲洗3-5 CV ，然后迅速用至少5 CV纯化水冲洗。
5、消毒灭菌
消毒灭菌是为了将柱子中的微生物污染降至最低。由于超螺旋质粒DNA亲和层析介质具有高耐碱性，可使用NaOH溶液作为消毒剂。NaOH溶液可有效去除病毒、细菌、 酵母及内毒素，运行成本极低。消毒步骤为：
5.1 使用结合缓冲液冲洗层析柱3-5 CV。
5.2 使用0.5 M NaOH溶液冲洗层析柱2-5 CV。
5.3 使用0.1 M NaOH溶液对层析柱浸泡30min（或者0.5 M NaOH浸泡15min）
5.4 用pH在7-8之间的结合缓冲液冲洗层析柱5-10 CV后即完成消毒过程。
注意事项： 层析柱污染程度严重时，使用混有30-40%丙醇的0.5 M NaOH进行清洗。高浓度NaOH或长时间NaOH处理会降低介质载量，消毒时要注意清洗浓度和时间。
6、保存
未使用的介质应当保存在4-30℃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。
已装好介质的层析柱应当使用20%乙醇或者2%苯甲醇浸泡密封，4-30℃保存，防止微生物污染。在使用前应当进行一次空载运行，并进行CIP。
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。
备注：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
Affinity Chromatography (AC) is a chromatographic technique that separates molecules based on specific biological affinities. When a sample passes through the stationary phase, the target biomolecule binds specifically to the immobilized ligand, while impurities flow through with the eluent, enabling the separation and purification of the biomolecule. Affinity chromatography is one of the most commonly used techniques for purifying proteins, enzymes, and other macromolecules, offering a simple and rapid separation process. It is also frequently used in the study of macromolecular structure and function.
This product is a thiophilic affinity chromatography medium prepared by coupling 2-mercaptopyridine to a highly rigid agarose matrix. Thiophilic affinity chromatography separates and purifies biomolecules based on electron donor-acceptor interactions, which are enhanced under high-salt conditions and weakened under low-salt conditions. This medium is primarily used for the purification of supercoiled plasmid DNA. During purification, supercoiled plasmid DNA adsorbs onto the medium, while open-circular plasmid DNA does not bind, achieving effective separation. The resulting high-quality supercoiled plasmid DNA is commonly used in gene therapy or as a DNA vaccine.
Aladdin Plasmid Agarose Resin is stored in 20% ethanol, 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 | Highly Rigid Agarose |
| Ligand | 2-Mercaptopyridine |
| Average Particle Size | 40 μm |
| Binding Capacity | ~3 mg supercoiled plasmid DNA / mL medium |
| Recommended Flow Rate | ＜300 cm/h |
| Recommended Pressure | ＜0.5 MPa (5 bar) |
| pH Stability¹ | 3-11 (long term); 2-13 (short term) |
| Chemical Stability | Stable in common aqueous buffers (e.g., 70% ethanol, 0.1 M NaOH, 1 M acetic acid) |
| Storage | In 20% Ethanol, 4-30°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 an appropriate amount of packing buffer to the exchanged medium to create a ~50% 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 10 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 1200 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 1-3 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 > 9,000.
3. Separation and Purification
3.1 Column Equilibration
Connect the column to the purification system. Equilibrate with Binding Buffer for more than 2-3 CV until the pH and conductivity of the column effluent match those of the Binding Buffer. Zero the UV detector after equilibration.
*Note: Binding Buffer: 100 mM Tris-HCl, 2.1 M (NH₄)₂SO₄, 10 mM EDTA, pH 7.5, filtered through 0.2 μm.*
3.2 Sample Loading
Load the sample containing supercoiled plasmid DNA (filtered through a 0.45 μm membrane) onto the column. The loading volume depends on the medium's binding capacity, the concentration of supercoiled plasmid DNA in the sample, and other chromatographic conditions. Samples treated for RNA removal can be loaded directly. Other samples may require salt and acid (or base) adjustment to match the conductivity and pH of the Binding Buffer before filtration (0.45 μm) and loading.
*Note: Recommended sample loading flow rate < 150 cm/h. Slower flow rates are needed for taller columns.*
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 280 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.
*Note: Elution Buffer: 100 mM Tris-HCl, 1.7 M (NH₄)₂SO₄, 10 mM EDTA, 0.3 M NaCl, pH 7.5, filtered through 0.2 μm.*
3.5 Cleaning and Storage
Wash the column with 3 CV of purified water, followed by 3-5 CV of 0.5 M NaOH. Then rinse with purified water until neutral pH is reached. Finally, rinse with 2-3 CV of 20% ethanol. Store the column at 4-30°C.
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. Common CIP methods:
4.1 Removing Denatured Proteins: Wash the column with 3-5 CV of 0.5 M NaOH, then immediately flush with at least 5 CV of Binding Buffer.
4.2 Removing Strongly Hydrophobic Proteins or Lipids: Wash the column with 3-5 CV of 20 mM PB, 30% Isopropanol, pH 7.5, then quickly flush with at least 5 CV of purified water.
5. Sanitization
Sanitization minimizes microbial contamination in the column. Due to its high alkali resistance, NaOH solution can be used as a sanitizing agent for this medium. NaOH effectively removes viruses, bacteria, yeast, and endotoxins at very low cost. Sanitization procedure:
5.1 Flush the column with 3-5 CV of Binding Buffer.
5.2 Flush the column with 2-5 CV of 0.5 M NaOH solution.
5.3 Soak the column in 0.1 M NaOH for 30 minutes (or 0.5 M NaOH for 15 minutes).
5.4 Flush the column with 5-10 CV of Binding Buffer (pH 7-8) to complete the sanitization process.
*Precautions: For heavily contaminated columns, use 0.5 M NaOH containing 30-40% propanol for cleaning. High NaOH concentrations or prolonged exposure can reduce medium binding capacity; pay attention to concentration and time during sanitization.*
6. Storage
Unused medium should be stored in a dry, ventilated, clean environment at 4-30°C, ensuring the container is tightly sealed. Do not freeze.
Packed columns should be stored submerged in 20% ethanol or 2% benzyl alcohol, sealed, at 4-30°C to prevent microbial growth. Before use, perform a blank run and CIP.
To prevent microbial growth due to ethanol evaporation, replace the storage solution with fresh solution every 3 months.
Note: 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
超螺旋DNA填料 | 超螺旋DNA琼脂糖凝胶
英文别名
Plasmin Select | Plasmid Capture 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
Plasmid 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 |
|---|---|---|
 ZJ25F1029074 |
分析证书 | P1492538 |
| ZJ25F1029073 |
分析证书 | P1492538 |
技术文档和文章
溶液计算器
常见问题
什么是「通过内毒素测试」？
「通过内毒素测试」表示该物料经过加工和检测，适用于特种工业加工。规格侧重于影响该应用性能的杂质——例如，电子应用的金属含量、电池应用的粒度、光学应用的折射率，或对水分敏感工艺的水分含量。
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品应如何储存？
请于2–8 °C条件下保存。请勿冷冻。冷冻可能对本品造成不可逆的损坏。
本产品如何运输？
本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
相关产品
耐碱 rProtein A 亲和层析介质,CAS:P405880
Dextrin 亲和层析介质 (MBP tag)-BioReagent, 通过内毒素测试, 50% v/v
Strep-tag II 标签亲和层析介质（耐生物素）
rProtein A/G 亲和层析介质-BioReagent, 通过内毒素测试, 50% v/v
rProtein G 亲和层析介质
Anti-Flag 亲和层析介质-BioReagent, 50% v/v
Anti-c-Myc 亲和层析介质
Anti-HA 亲和层析介质
谷胱甘肽亲和层析介质 (GST tag)
UltraBio™ His-tag 纯化树脂
Ni-TED 亲和层析介质 6FF (His tag) -BioReagent,用于蛋白分析,50% v/v；45~165 μm
Ni-IDA 亲和层析介质 (His tag)-BioReagent, 用于蛋白分析, 25-35 mg His-Tagged protein/mL wet gel; 75 μm
Strep-tag II 标签亲和层析介质-BioReagent, 50% v/v
链霉亲和素亲和层析介质-BioReagent, 50% v/v
Anti-GFP 亲和层析介质-BioReagent, 50% v/v
Anti-RFP 亲和层析介质-BioReagent, 50% v/v
rProtein L 亲和层析介质-BioReagent, 通过内毒素测试, 50% v/v
生物素亲和层析介质-BioReagent, 通过内毒素测试, 50% v/v
Con A 亲和层析介质-BioReagent, 通过内毒素测试, 50% v/v
肝素亲和层析介质-BioReagent, 通过内毒素测试, 50% v/v
内毒素去除亲和层析介质-BioReagent, 50% v/v
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
Copyright © 2023至今 上海阿拉丁生化科技股份有限公司 保留所有权利。
危险品化学品经营许可证（带存储）