储存缓冲液：20% (v/v) Ethanol
本产品是基于高交联琼脂糖基球的预活化层析介质，具有良好的物理和化学稳定性，能耐受不同温度、pH以及多种化学试剂。本产品能够快速结合含巯基的多肽、蛋白或者其他生物配体，可以与巯基形成稳定的硫醚键，从而将配体固定到介质上，进而利用抗原抗体的特异性反应对免疫血清中的抗体进行高效纯化，是多克隆抗体生产中不可缺少的纯化介质。
阿拉丁巯基蛋白亲和层析介质储存在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
| 4%高交联琼脂糖
| 偶联量
| 3 - 20 mg/mL
| 平均粒径
| ~90 μm
| 最大运行流速①
| 500 cm/h
| 工作温度
| 4 - 40°C
| 耐压
| 0.3 MPa
| 化学稳定性
| 常用水相缓冲液 | 储存
| 20%乙醇，2~8℃
| 保质期
| 18个月 |
|
注①：300/500层析柱，柱床高度20cm，纯化水，20℃。
使用说明
1、缓冲液配制
偶联缓冲液：50 mM Tris-HCl，5 mM EDTA，pH 8.5.
封闭缓冲液：50 mM Tris-HCl，5 mM EDTA，50 mM L-半胱氨酸，pH 8.5
2、偶联步骤
2.1 用偶联缓冲液替换介质保存液，然后冲洗介质至白色，抽干介质；
2.2 用偶联缓冲液配制一定浓度的配体溶液（0.5 - 10 mg/mL），浓度根据所使用的配体而定；
2.3 将介质与配体溶液1:1混合；
2.4 均匀搅拌混合物或置于摇床中震荡，使介质充分悬浮，禁止使用磁力搅拌器，室温下反应0.5- 2h；
2.5 用偶联缓冲液洗去多余配体；
2.6 将介质转移至封闭液中，震荡15min；
2.7 最后用纯化水清洗介质。
3、测定偶联效率
3.1 测定偶联配体溶液于280 nm处的吸光度为A1。
3.2 取偶联结束后的配体溶液测定280 nm处的吸光度为A2。
3.3 公式计算
偶联效率=[(A1*V1-A2*V2)/(A1*V1)] *100%
A1 = A₂₈₀（偶联前配体溶液）
V1 = 参与偶联的配体溶液体积
A2 = A₂₈₀（反应后的收集液）
V2 = 配体偶联后洗涤液的体积
4、装柱
材料：层析柱、偶联后层析介质、装柱缓冲液（纯化水或平衡缓冲液）
4.1 根据计划装填的层析柱的柱体积计算需要的填料量。需要的沉降介质体积=柱体积×压缩比
4.2 将介质悬液充分摇匀后量取上述方法计算所得的体积，转移到容器中后，加入约3倍填料体积的纯化水，填料沉降后，去上清，（也可倒入漏斗，抽去液体），重复3次，每次加纯水时需用玻璃棒或者搅胶棒搅拌，以便更好地清洗掉保存液。
4.3 装柱胶悬液准备：将清洗好的介质转移到烧杯或其它适当的容器中，加入装柱缓冲液至胶悬液浓度为50 - 75%，搅拌均匀。
4.4 取清洗干净的层析柱，用纯化水，通过层析柱下端口排净下筛网内气泡，在柱子底部保留约1- 2cm高的液体，关闭或拧紧下堵头，调整柱子使其垂直于地面。
4.5 将搅匀后的胶悬液一次性缓慢倒入层析柱内（如有需要可使用装柱器），注意不要带入气泡。
4.6 装柱类型
对于实验室用层析柱：用装柱缓冲液将装柱器加满，将装柱器与层析系统连接，开启流速，排空软管中气泡，关闭流速，再拧紧装柱器上盖。待填料完全自然沉降或以30-60 cm/h流速冲至胶面稳定后，去掉装柱器，装上上柱头，将柱头下降至胶面上约1cm的位置，开启流速或者恒压压柱至胶面清晰稳定，标记胶面稳定时的柱高。稍微放松柱头密封圈，下压柱头至标记位置下面约0.5 cm， 旋紧柱头密封圈，装柱完成；
对于工业用层析柱：倒入后用搅胶棒再次搅匀， 让介质自然沉降，直至悬液上有大约1-2 cm澄清液。安装上柱头，将上柱头与层析系统连接。调节上柱头使其下降到与澄清液接触，待密封圈全 部浸入澄清液后拧紧密封圈。确保层析柱顶部阀门打开情况下，慢慢下移上柱头至所有气泡排净。待填料完全自然沉降或以30 - 60 cm/h 流速冲至胶面稳定后，将柱头下降至胶面上约1 cm的位置，开启流速或恒压压柱至胶面清晰稳定，标记胶面稳定时的柱高，稍微放松柱头密封圈，下压柱头至标记位置下面约0.5 cm，旋紧柱头密封圈，或者按照填料压缩比下压上柱头至正确位置，装柱完成。
5、柱效测定和评价
柱效测定可以通过以下两种方法进行检测：
| 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%柱体积
| 0.8 M NaCl，1.0%柱体积
| | 流速 | 30 cm/h
| 30 cm/h
| | 检测信号 | UV-280 nm
| 电导 |
|
根据理论塔板高度（Height equivalent to a theoretical plate，HETP）、理论塔板数（number of theoretical plates，N）和非对称因子（Asymmetry factor，As）可有效的评价层析柱的性能。
公式如下：
- HETP = L / N
- N = 5.54 × (Vʀ / Wₕ/₂)²
- As = b / a
Vʀ= 进样点到色谱峰极大点的距离，单位为毫米（mm）；
Wₕ/₂ = 半高峰宽，单位为毫米（mm）；
L = 柱高，单位为厘米（cm）；
a = 峰高为10%时所对应的左半峰宽，单位为毫米（mm）；
b = 峰高为10%时所对应的右半峰宽，单位为毫米（mm）。
柱效评估标准
柱效良好的层析柱，其理论塔板高度HETP的数值应小于介质平均颗粒大小的三倍；
检测峰形必须对称，非对称因子数值As应在0.8 1.5 之间，越接近1柱效越好；
如柱效检测不达标，则需重新装柱。
6、样品预处理
为了保证层析介质使用寿命，样品在进入到层析柱前应使用0.22或0.45 μm滤膜过滤。并确保样品的 pH 值和离子强度与平衡缓冲液相同。
6.1 结合
吸附取决于样品浓度、流速、pH、缓冲液成分和温度等参数。平衡缓冲液的组成应根据靶蛋白和配体的性质进行优化。
6.2 洗脱
可以通过改变缓冲液的pH值、离子强度或极性，或添加变性剂来实现洗脱。应针对不同的样品和配体优化洗脱条件，以获得最大的纯度。
6.3 再生
再生的条件取决于所偶联的配体。一般可用洗脱缓冲液洗涤再生，随后用平衡缓冲液重新平衡。
6.4 在位清洗
在位清洗（CIP）可去除再生后仍留在层析柱中的杂质，如脂质、内毒素、核酸和沉淀或变性蛋白质。定期 CIP 可有效保护介质，一般建议在正常使用期间，至少每5个周期清洗一次层析柱。但对于污染严重的介质， 必须立即运行CIP。CIP 条件取决于偶联配体，如果配体稳定性好，可以使用6 M盐酸胍、8 M尿素甚至0.1 - 0.5 M NaOH清洗。
6.5 工艺放大
在实验室规模确定纯化方案后，该工艺可以放大到中试和生产规模。工艺放大通常通过保持柱床高度和线性流速恒定，同时增加层析柱直径和体积流速来实现。 由于实验室进行条件优化时，为了节省样品与缓冲液，通常使用柱体积较小的层析柱。有一部分参数优化时，如动态吸附载量，可以使用规格更短的层析柱。只要保证保留时间不变，目标分子的结合能力就保持不变。但一些其他因素，如关键杂质的清除，可能随着柱床高度的变化而变化，需要在最终生产使用的层析柱中进行验证。工艺放大的推荐方案如下：
- 根据生产需求选择柱体积。如果柱床高度改变， 重新验证纯化方案。
- 跟据实际情况选择层析柱直径。然后根据保留时间，确定柱床高度。注意，在流速恒定的情况下， 反压随柱床高度的增加而成比例增加。
- 样品浓度和洗脱条件保持不变。
常见问题及解决方法
1、层析柱反压过高
- 检查泵和收集容器之间的所有阀门是否完全打开。
- 检查所有阀门是否清洁、堵塞。
- 检查使用中的设备是否在层析柱前后产生反压（例如，阀门和流路单元尺寸不正确）。
- 执行CIP以去除介质中紧密结合的杂质。
- 根据层析柱使用说明书检查层析柱部件，如过滤器、筛网等。
2、色谱结果未达预期
- 检查速度/信号记录仪是否出现故障。
- 检查流速。
- 检查缓冲液。
- 检查柱头与胶面之间是否有间隙，或在应用前样品是否混合均匀。
- 检查柱效。
- 检查样品的预处理是否有变化。
3、污染
- 检查连接和预过滤器。
- 检查使用过的液体，如缓冲液、样品组分等。
- 检查层析柱是否正确消毒。
4、产生气泡
- 检查缓冲液是否与层析柱温度相同。
- 检查连接是否松动，阀门是否漏水。
- 如果空气已进入层析柱，则应重新装柱。如果只有少量空气在柱床顶部，或纯化仪与上柱头之间 管路，可以通过平衡液反向冲洗来去除气泡。在此之后，重新检测柱效，并与初始结果进行比较。
储存和运输
未开封的产品，2 - 8℃储存，有效期为18个月。
使用后的介质可以保存在20%乙醇溶液中，2-8℃条件下保存，请勿冷冻介质。为了防止乙醇挥发和微生物滋生，建议定期更换新鲜的保存液。
巯基蛋白亲和层析介质的推荐储存或运输温度为2-8°C，也可常温短途运输，常温短途运输不会影响介质性能。
This product is a pre-activated chromatography medium based on a highly cross-linked agarose matrix, offering excellent physical and chemical stability. It can withstand various temperatures, pH levels, and numerous chemical reagents. This medium rapidly binds thiol-containing peptides, proteins, or other biological ligands, forming stable thioether bonds with thiol groups, thereby immobilizing the ligand onto the matrix. It is subsequently used for the efficient purification of antibodies from immune serum via antigen-antibody specific interactions, making it an essential purification medium in polyclonal antibody production.
Aladdin SulfoLink Coupling Agarose Resin is stored in 20% ethanol, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Specification
| Matrix
| 4% Highly Cross-linked Agarose
| Coupling Capacity
| 3 - 20 mg/mL
| Average Particle Size
| ~90 μm
| Max Flow Rate¹
| 500 cm/h
| Operating Temperature
| 4 - 40°C
| Pressure Tolerance
| 0.3 MPa
| Chemical Stability
| Stable in common aqueous buffers | Storage
| In 20% Ethanol, 2~8°C
| Shelf Life
| 18 months |
|
Note1: Determined using a 300/500 chromatography column, 20 cm bed height, purified water, 20°C.
Instructions for Use
1. Buffer Preparation
Coupling Buffer: 50 mM Tris-HCl, 5 mM EDTA, pH 8.5.
Blocking Buffer: 50 mM Tris-HCl, 5 mM EDTA, 50 mM L-Cysteine, pH 8.5.
2. Coupling Procedure
2.1 Replace the storage solution of the medium with Coupling Buffer. Wash the medium until it appears white. Drain the medium.
2.2 Prepare a ligand solution (0.5 - 10 mg/mL) in Coupling Buffer. The concentration depends on the specific ligand used.
2.3 Mix the medium with the ligand solution at a 1:1 ratio.
2.4 Stir the mixture evenly or place it on a shaker to ensure the medium is fully suspended. Do not use a magnetic stirrer. React at room temperature for 0.5 - 2 hours.
2.5 Wash away excess ligand using Coupling Buffer.
2.6 Transfer the medium to the Blocking Buffer and shake for 15 minutes.
2.7 Finally, wash the medium with purified water.
3. Determining Coupling Efficiency
3.1 Measure the absorbance of the ligand solution before coupling at 280 nm (A₁).
3.2 Measure the absorbance of the ligand solution after coupling (the collected wash solution) at 280 nm (A₂).
3.3 Calculation Formula:
Coupling Efficiency (%) = [(A₁ × V₁ - A₂ × V₂) / (A₁ × V₁)] × 100%
* A₁ = A₂₈₀ (Ligand solution before coupling)
* V₁ = Volume of ligand solution used for coupling
* A₂ = A₂₈₀ (Collected solution after coupling reaction)
* V₂ = Volume of the post-coupling wash solution
4. Column Packing
Materials: Chromatography column, coupled chromatography medium, packing buffer (purified water or equilibration buffer)
4.1 Calculate the required amount of medium based on the planned column volume. Required settled medium volume = Column Volume × Compression Ratio.
4.2 Resuspend the medium slurry thoroughly and measure the volume calculated above. Transfer to a container, add about 3 times the medium volume of purified water. After the medium settles, remove the supernatant (or pour into a funnel and aspirate the liquid). Repeat this washing step 3 times. Stir with a glass rod or spatula each time when adding water to ensure complete removal of the storage solution.
4.3 Preparation of Packing Slurry: Transfer the washed medium to a beaker or suitable container. Add packing buffer to achieve a slurry concentration of 50 - 75%. Mix evenly.
4.4 Take a cleaned chromatography column. Use purified water to purge air bubbles from the bottom screen via the lower port. Retain about 1-2 cm of liquid at the column bottom. Close or tighten the bottom end cap. Position the column vertically.
4.5 Pour the well-mixed slurry into the column slowly and in one go (use a packing reservoir if needed). Avoid introducing air bubbles.
4.6 Column Packing Type
* For Lab-scale Columns: Fill the packing reservoir with packing buffer. Connect the reservoir to the chromatography system, start the flow to purge air bubbles from the tubing, then stop the flow and tighten the reservoir cap. After the medium has settled completely naturally or been compressed at 30-60 cm/h until the bed is stable, remove the reservoir and attach the top adaptor. Lower the adaptor to about 1 cm above the bed surface. Start the flow or apply constant pressure until the bed surface is clear and stable. Mark the bed height at stability. Loosen the adaptor seal slightly, lower the adaptor to about 0.5 cm below the marked position, and tighten the seal. Packing is complete.
* For Industrial-scale Columns: After pouring, mix the slurry again with a stirrer. Let the medium settle naturally until there is about 1-2 cm of clear supernatant. Install the top adaptor and connect it to the chromatography system. Adjust the top adaptor downward until it contacts the supernatant. After the seal is fully immersed, tighten it. With the top valve open, slowly lower the adaptor to expel all air bubbles. After the medium has settled completely naturally or been compressed at 30-60 cm/h until the bed is stable, lower the adaptor to about 1 cm above the bed surface. Start the flow or apply constant pressure until the bed surface is clear and stable. Mark the bed height at stability. Loosen the adaptor seal slightly, lower the adaptor to about 0.5 cm below the marked position, and tighten the seal. Alternatively, lower the adaptor to the correct position based on the medium's compression ratio. Packing is complete.
5. Column Efficiency Testing and Evaluation
Column efficiency can be tested using the following two methods:
| Method | Acetone Test | NaCl Test
| | Equilibration Buffer | Purified Water
| 0.4 M NaCl
| | Sample | 1% Acetone in water, 1.0% CV | 0.8 M NaCl, 1.0% CV
| | Flow Rate | 30 cm/h
| 30 cm/h
| | Detection Signal | UV-280 nm
| Conductivity |
|
Column performance can be effectively evaluated based on the Height Equivalent to a Theoretical Plate (HETP), the Number of Theoretical Plates (N), and the Asymmetry Factor (As).
Formulas:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ/₂)²
As = b / a
Where:
Vʀ = Distance from injection point to peak maximum (mm)
Wₕ/₂ = Peak width at half height (mm)
L = Column height (cm)
a = Width of the left side of the peak at 10% peak height (mm)
b = Width of the right side of the peak at 10% peak height (mm)
Column Efficiency Evaluation Criteria:
For a well-packed column, the HETP value should be less than three times the average particle size of the medium.
The peak shape must be symmetrical, with an Asymmetry Factor (As) between 0.8 and 1.5. A value closer to 1 indicates better column efficiency.
If the column efficiency test fails, the column must be repacked.
6. Sample Preparation
To ensure the longevity of the chromatography medium, the sample should be filtered through a 0.22 or 0.45 μm membrane before application to the column. Ensure the sample's pH and ionic strength match those of the equilibration buffer.
6.1 Binding: Binding depends on parameters such as sample concentration, flow rate, pH, buffer composition, and temperature. The composition of the equilibration buffer should be optimized based on the properties of the target protein and the ligand.
6.2 Elution: Elution can be achieved by changing the buffer pH, ionic strength, or polarity, or by adding denaturants. Elution conditions should be optimized for different samples and ligands to achieve maximum purity.
6.3 Regeneration: Regeneration conditions depend on the coupled ligand. Generally, washing with the elution buffer followed by re-equilibration with the equilibration buffer is sufficient.
6.4 Cleaning In Place (CIP): CIP removes impurities that remain on the column after regeneration, such as lipids, endotoxins, nucleic acids, and precipitated or denatured proteins. Regular CIP effectively protects the medium. It is generally recommended to perform CIP at least every 5 cycles during normal use. For severely contaminated media, CIP must be run immediately. CIP conditions depend on the coupled ligand's stability. If the ligand is stable, 6 M guanidine hydrochloride, 8 M urea, or even 0.1 - 0.5 M NaOH can be used for cleaning.
6.5 Process Scale-Up: After establishing the purification protocol at laboratory scale, the process can be scaled up to pilot and production scales. Scale-up is typically achieved by maintaining constant bed height and linear flow rate while increasing the column diameter and volumetric flow rate. Since smaller columns are often used for condition optimization in the lab to save sample and buffer, some parameters (e.g., dynamic binding capacity) can be optimized using shorter columns. As long as the residence time remains constant, the binding capacity for the target molecule remains unchanged. However, other factors, such as the clearance of critical impurities, may change with bed height and need verification in the final production column. The recommended scale-up strategy is as follows:
* Select the column volume based on production needs. If the bed height changes, revalidate the purification protocol.
* Choose the column diameter based on the actual situation. Then determine the bed height based on the residence time. Note: At a constant flow rate, backpressure increases proportionally with bed height.
* Keep sample concentration and elution conditions unchanged.
Troubleshooting
High Column Backpressure:
Check if all valves between the pump and the collection vessel are fully open.
Check all valves for cleanliness and blockage.
Check if equipment in use (e.g., valves, flow path units of incorrect size) is generating backpressure before or after the column.
Perform CIP to remove tightly bound impurities from the medium.
Check column components (filters, screens, etc.) according to the column's manual.
Chromatography Results Not as Expected:
Check the speed/recorder for malfunctions.
Check the flow rate.
Check the buffers.
Check for gaps between the adaptor and the bed surface, or if the sample was mixed homogeneously before application.
Check the column efficiency.
Check for changes in sample preparation.
Contamination:
Check connections and pre-filters.
Check the liquids used (buffers, sample components, etc.).
Check if the column was sanitized correctly.
Air Bubbles:
Check if the buffer temperature matches the column temperature.
Check for loose connections or leaking valves.
If air has entered the column, it should be repacked. If only a small amount of air is at the top of the bed or in the tubing between the purifier and the top adaptor, it can sometimes be removed by reverse flushing with equilibration buffer. Afterwards, re-test the column efficiency and compare it with the initial results.
Storage and Transportation
Unopened products should be stored at 2 - 8°C with a shelf life of 18 months.
Used media can be stored in 20% ethanol solution at 2-8°C. Do not freeze the medium. To prevent ethanol evaporation and microbial growth, replace the storage solution periodically with fresh solution.
The recommended storage and transportation temperature for the Thiol Protein Affinity Chromatography Medium is 2-8°C. Short-term transportation at ambient temperature is also acceptable and will not affect medium performance.
ZJ25F1029234
危险品化学品经营许可证（带存储）