硫酸葡聚糖亲和层析介质（低硫/蛋白纯化型）是将硫酸葡聚糖（Dextran Sulfate）键合在高刚性琼脂糖微球上形成的一种亲和层析分离介质。硫酸葡聚糖又称右旋糖酐硫酸酯，是天然多糖右旋糖酐的合成衍生物，具有与肝素类似的的生物活性，是一种非动物源的仿肝素配基，填料产品被称为仿肝素亲和填料。
本产品可用于蛋白的亲和分离、纯化，例如血浆蛋白。同时，硫酸酯是一种优良的耐盐型阳离子交换配基，也可作为阳离子交换填料，对溶菌酶的结合量大于60 mg/mL，且在50~150 mM NaCl盐浓度下仍结合良好。
阿拉丁硫酸葡聚糖亲和层析介质（低硫/蛋白纯化型）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 | 高刚性琼脂糖 | 配基 | 硫酸葡聚糖，含硫量40~80 μmol/mL | 粒径范围 ① | 30~100 μm | 平均粒径 | ~60 μm | 结合能力 ② | ≥60 mg lysozyme/mL | 推荐工作流速 | 60~300 cm/h | 最大流速与压力 ③ | 1500 cm/h，0.3 MPa | 使用pH | 4~13（长期稳定性），3~14（CIP等短时间操作） | 化学稳定性 | 在常用的水相缓冲液、0.5 mol/L氢氧化钠、8 mol/L尿素、30%异丙醇和70%乙醇等体系中稳定。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 3年 |
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注：
① 90%体积以上的微球在此粒径范围；
② 测试方法为孵育2小时，该结果与DBC10%接近;
③ 10 cm 柱高下的最大测试流速。
使用说明
1、色谱柱装填
以下阐述与层析系统连接时，填料的色谱柱装填方法。
（1）所有需要用到的材料的温度要与色谱操作的温度一样，液体最好做脱气处理。装柱液一般为纯水，也可含10~100 mM NaCl。
（2）填料用量计算：通过沉降定量填料体积，需要的沉降填料体积=柱体积×压缩比（也称压缩因子）。沉降体积是指填料在20%乙醇保存液中自然沉降完全读取的稳定体积。硫酸葡聚糖亲和层析介质（低硫/蛋白纯化型）的压缩比为~1.10。为使达到压缩比，可通过柱头下压的方法，也可以通过高流速压柱。
（3）填料清洗：将填料悬液充分摇匀后量取相应体积，抽去液体，并用约3 倍填料体积的纯化水洗涤，重复3 次，以除去保存液。
（4）装柱填料悬液准备：将填料转移到适当的容器中，加入适量的装柱液，制成50~75 v%的装柱填料悬液，使用前搅匀。
（5）装填前准备：在清洗干净的层析柱下端加入装柱液，以除去下垫片及层析柱下端的空气，在柱内保留少量的蒸馏水，拧紧下堵头，调整柱子使其垂直于地面。
（6）装填：将搅匀后的填料悬液一次性缓慢倒入层析柱内（必要时使用装柱器），
为避免引入气泡，应使之沿层析柱内壁自然流下。将所有填料加入后，用装柱液将装
柱器加满，拧紧装柱器上盖，将柱子与层析系统连接。
（7）压柱：使柱内填料自然沉降（或50~150 cm/h 的低流速下沉降），待填料沉降完全后（填料与液体的界面清晰），去除装柱器，装上上柱头，并将柱头下降至界面处；通过高流速（推荐流速见下表，注意柱压不超过0.3 MPa），继续压柱至界面清晰稳定，标记界面稳定时的柱高。停泵，打开柱头上的阀门/堵头，关闭柱底的阀门/堵头，下压柱头至标记位置下方与压缩比对应的位置，旋紧柱头，装柱完成。装柱完成后，需用高流速平衡柱内的填料。
装柱条件 | 硫酸葡聚糖亲和层析介质（低硫/蛋白纯化型） | 压缩比 | 1.10 | 装柱流速 | 600~1500 cm/h |
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2、柱效测定和评价
完成装柱后、使用前可通过柱效测定和评价可以确认层析柱装填质量。柱效通常用理论塔板高度（HETP）和非对称因子（As）来评价。柱效测定可以采用丙酮或者NaCl作为样品进行，按照下表配制样品溶液和流动相。
样品 | 1.0%丙酮 | 0.8~1.0 M NaCl | 样品体积 | 1.0%柱体积 | 1.0%柱体积 | 流动相 | 纯水 | 0.4 M NaCl | 流速 | 30 cm/h | 30 cm/h | 检测器 | UV-280 nm | 电导 |
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根据UV或者电导率曲线计算理论塔板高度（HETP）、理论塔板数（N）和非对称因子（As），公式如下：
HETP = L / N
N = 5.54 × (VR / Wh )2
As = a / b
其中：L为柱高； 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。
一般来说，HETP的数值应小于填料平均粒径的三倍（即HETP/D50<3，D50为填料的平均粒径），As应在0.8~1.5之间。
3、平衡与上样（Equilibration & Loading）
在上样前，可用初始缓冲液A在操作流速下平衡层析柱，观察检测器的变化，直到电导、pH等参数不变。根据应用不同，本产品的缓冲液体系可为磷酸盐、柠檬酸盐和Tris-HCl等，pH为4~9居多，例如10 mM PB + 150 mM NaCl，pH7.5。
样品通常溶于上述缓冲液A，或者将样品溶液进行换液处理，置换成缓冲液A体系。上样量可按“mg目标蛋白/mL填料”来设定，通常为DBC10%的50~80%，除了DBCR，也可以测试上样流穿液中的目标蛋白确定上样量。
4、洗脱 （Elution）
洗脱过程则可以进一步提高缓冲液A中的盐浓度，添加0.5~2 M的NaCl可将目的蛋白有效洗脱。在前期工艺筛选时建议用线性梯度洗脱以确定合适的洗脱浓度，在工艺优化和确定时建议用阶跃梯度洗脱，以节省洗脱液体积和提高效率。
5、再生（Regeneration）和在位清洗（CIP）
通常上述的再生过程可将填料彻底清洗。若发生柱压升高，或为了避免交叉污染，可采用以下溶剂进行再生与在位清洗：1~2 mol/L NaCl，0.5 mol/L NaOH、70%乙醇或30%异丙醇等。在位清洗可有效去除介质上的杂质，反向效果更佳。清洗后需用3~5 柱体积的纯水除去上述溶剂。
CIP 过程 | 目的 | 1~2 mol/L NaCl，0.5~2 CV；水，3~5 CV。 | 除去通过离子相互作用结合的杂质 | 0.5 mol/L NaOH，1~2 h的接触时间，若除去内毒素则需更长时间；缓冲液A，5~10 CV；水，3~5 CV。 | 去除蛋白沉淀、疏水性蛋白或去除热源等其他杂质 | 30%异丙醇或70%乙醇，5~10 CV；水，3~5 CV。 | 去除紧密结合的脂蛋白和脂质体等 |
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6、填料保存
填料的初始保存液为20%乙醇，使用过后可继续用相同的保存液，也可在20%乙醇的基础上添加0.1~1 M NaCl。保存温度在2~8 ℃为宜，不可冻存。
Dextran Sulfate Affinity Resin (Low Sulfur / For Protein Purification)is an affinity chromatography separation medium formed by covalently coupling dextran sulfate onto highly rigid agarose microspheres. Dextran sulfate is a synthetic derivative of the natural polysaccharide dextran. It possesses biological activity similar to heparin and is a non-animal-derived heparin-mimetic ligand. The resin product is referred to as heparin-mimetic affinity resin.
This product can be used for the affinity separation and purification of proteins, such as plasma proteins. Meanwhile, sulfate ester is an excellent salt-tolerant cation exchange ligand, so it can also serve as a cation exchange resin. Its binding capacity for lysozyme is greater than 60 mg/mL, and it maintains good binding at NaCl concentrations ranging from 50 to 150 mM.
Aladdin Dextran Sulfate Affinity Resin (Low Sulfur / For Protein Purification) is stored in 20% ethanol, with a gel-to-protectant volume ratio of 1:1. The product specification indicates the actual gel volume.
Item | Specification | Matrix | Highly rigid agarose | Ligand | Dextran sulfate, sulfur content 40–80 μmol/mL | Particle size range | ① 30–100 μm | Average particle size | ~60 μm | Binding capacity | ② ≥60 mg lysozyme/mL | Recommended working flow rate | 60–300 cm/h | Maximum flow rate and pressure | ③ 1500 cm/h, 0.3 MPa | Working pH | 4–13 (long-term stability), 3–14 (short-term operation such as CIP) | Chemical stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 30% isopropanol, 70% ethanol, etc. | Storage | 20% ethanol, 2–8 °C | Shelf life | 3 years |
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Notes:
①More than 90% of the microspheres by volume fall within this particle size range;
② Test method: incubation for 2 hours; the result is close to DBC<sub>10%</sub>;
③ Maximum tested flow rate at a column height of 10 cm.
Instructions for Use
1. Column Packing
The following describes the column packing method for the resin when connected to a chromatography system.
(1)The temperature of all materials used shall be consistent with that of the chromatography operation; liquids are preferably degassed. The packing solution is generally pure water, which may also contain 10–100 mM NaCl.
(2) Resin volume calculation: Quantify the resin volume by sedimentation. Required settled resin volume = column volume × compression ratio (also called compression factor). Settled volume is the stable volume read after the resin settles completely naturally in 20% ethanol storage solution. The compression ratio of Dextran Sulfate Affinity Resin (Low Sulfur / For Protein Purification) is ~1.10. To achieve the compression ratio, compress the column by lowering the top adapter or at a high flow rate.
(3) Resin washing: Shake the resin suspension thoroughly, measure the required volume, remove the liquid, and wash with approximately 3 column volumes of pure water; repeat 3 times to remove the storage solution.
(4) Preparation of packing suspension: Transfer the resin to a suitable container, add an appropriate amount of packing solution to prepare a 50–75 v% resin suspension, and mix well before use.
(5) Pre-packing preparation: Add packing solution to the bottom of the cleaned chromatography column to remove air from the lower frit and the column bottom, leave a small amount of distilled water in the column, tighten the lower plug, and adjust the column to be vertical to the ground.
(6) Packing: Pour the well‑mixed slurry slowly into the column in one go (use a packing funnel if needed).To prevent air bubbles, let it run down gently along the inner wall of the column.Once all slurry has been added, fill the packing funnel completely with packing buffer, fasten the top lid of the packing funnel, and connect the column to the chromatography system.
(7) Compression: Let the resin settle naturally in the column (or settle at a low flow rate of 50–150 cm/h). After complete sedimentation (clear interface between resin and liquid), remove the packing reservoir, install the top adapter, and lower the adapter to the interface; continue compression at a high flow rate (see the table below for recommended flow rate; note that column pressure does not exceed 0.3 MPa) until the interface is clear and stable, and mark the column height at stable interface. Stop the pump, open the valve/plug on the top adapter, close the valve/plug at the column bottom, lower the adapter to the position corresponding to the compression ratio below the marked position, tighten the adapter, and packing is completed. After packing, equilibrate the resin in the column at a high flow rate.
Packing Condition | Dextran Sulfate Affinity Resin (Low Sulfur / For Protein Purification) | Compression ratio | 1.10 | Packing flow rate | 600–1500 cm/h |
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2. Column Efficiency Test & Evaluation
Column efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.
Item | 1.0% Acetone | 0.8–1.0 M NaCl | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
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Calculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:
- HETP = L / N
- N = 5.54 × (Vr / Wh)2
- As = a / b
Where:L = column height; V<sub>r</sub> = retention volume; W<sub>h</sub> = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration & Loading
Before loading, equilibrate the column with starting buffer A at the operating flow rate and monitor the detector until parameters such as conductivity and pH remain constant. Depending on the application, buffer systems for this product can be phosphate, citrate, Tris-HCl, etc., mostly at pH 4–9, e.g., 10 mM PB + 150 mM NaCl, pH 7.5.
Samples are usually dissolved in the above buffer A, or the sample solution is buffer-exchanged into buffer A. Loading amount can be set as “mg target protein/mL resin”, typically 50–80% of DBC<sub>10%</sub>. In addition to DBC<sub>r</sub>, loading amount can also be determined by testing the target protein in the flow-through.
4. Elution
Elution can be performed by increasing the salt concentration in buffer A. Adding 0.5–2 M NaCl effectively elutes the target protein. Linear gradient elution is recommended during initial process screening to determine the appropriate elution concentration, while step gradient elution is recommended for process optimization and finalization to save eluent volume and improve efficiency.
5. Regeneration and Clean-in-Place (CIP)
The above regeneration process usually cleans the resin thoroughly. If column pressure increases or to avoid cross-contamination, the following solvents can be used for regeneration and CIP: 1–2 mol/L NaCl, 0.5 mol/L NaOH, 70% ethanol, 30% isopropanol, etc. CIP effectively removes impurities from the medium, with better results in reverse flow. After cleaning, remove the above solvents with 3–5 column volumes of pure water.
CIP Procedure | Purpose | 1–2 mol/L NaCl, 1–2 CV; water, 3–5 CV | Removes impurities bound by ionic interactions | 0.5 mol/L NaOH, 1–2 h contact time (longer for endotoxin removal); buffer A, 5–10 CV; water, 3–5 CV | Removes protein precipitates, hydrophobic proteins, or other impurities such as pyrogens | 30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV | Removes tightly bound lipoproteins, liposomes, etc. |
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6. Resin Storage
The initial storage solution for the resin is 20% ethanol. After use, it can still be stored in 20% ethanol. The suitable storage temperature is 2–8 °C; do not freeze.
ZJ26F0636587
危险品化学品经营许可证（带存储）