红色亲和层析介质是活性红120（又称普施安红HE-3B）活性染料键合在琼脂糖微球上得到的亲和填料。活性红120是多环染料，与NADP+具有结构类似性，可用于纯化核苷酸依赖性酶、脂蛋白、乳酸脱氢酶、纤溶酶原、肽、激素等的纯化。除了作为亲和填料使用，本产品配基含芳香环和阴离子，也通过静电作用或者疏水相互作用进行非亲和纯化。
阿拉丁红色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 | 6%交联琼脂糖 | 配基 | 活性红120，~2 μmol/mL | 粒径范围 ① | 45~165 μm | 平均粒径 | ~90 μm | 动态结合载量 ② | ~15 mg BSA/mL | 推荐工作流速 | 60~300 cm/h | 最大流速与压力 ③ | 1500 cm/h，0.5 MPa | 使用pH | 4~8.5（推荐的工作pH）， 3~12（长期稳定）；2~14（短期稳定） | 化学稳定性 | 在以下溶液中稳定：常用的水相缓冲液、0.5 mol/L氢氧化钠、8 mol/L尿素、 6 mol/L 盐酸胍、70%乙醇。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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注：
①90%体积以上的微球在此粒径范围；
②DBC 10% 测试条件为：10%流穿，6 min停留时间；
③10 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。
（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。红色亲和层析介质的压缩比为1.15。为使达到装柱比，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，抽滤除去液体，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，在柱内保留少量的装柱液，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 | 红色亲和层析介质 | 压缩比 | 1.15 | 装柱流速 | 600 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，需用缓冲液A在操作流速下平衡层析柱，该过程通常需要5~10个柱体积的缓冲液A，以电导、pH等检测信号参数不变且与缓冲液A对应为准。可以选择磷酸盐、Tris-HCl等常见的缓冲体系。
上样量可按“mg目标蛋白/mL填料”来设定，通常为DBC10%的50~80%，例如红色亲和层析介质的动态结合载量为~15 mg/mL，纯化时的上样量可为7.5~12 mg/mL。除了DBC10%，也可以测试上样流穿液中的目标蛋白确定上样量。上样前需要对样品进行离心（10000 g以上）、过滤（0.22或0.45 μm）处理，以免堵塞色谱柱。
上样后需要3~10个柱体积的缓冲液A再平衡层析柱。
4、洗脱（Elution）
通常在缓冲液A中添加3 M NaCl或者2 M KCl可将目标蛋白洗脱。
5、再生（Regeneration）
纯化后可用弱酸和弱碱性缓冲液交替冲洗色谱柱3~4次，进行填料再生，例如0.1mol/L NaAc + 0.5mol/L NaCl（pH4.5）和0.1mol/L Tris·HCl+0.5mol/L NaCl（pH8.5）。然后用平衡缓冲溶液平衡。
6、在位清洗（CIP）
通常上述的洗脱和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在位清洗：0.1~0.5 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 | 0.1~0.5 mol/L NaOH，~0.5 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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7、填料保存
填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇并添加0.5 M NaCl保存。保存温度在2~8℃为宜，不可冻存。
Red Sepharose 6FF is an affinity filler prepared by immobilizing Reactive Red 120 (also known as Procion Red HE-3B) reactive dye onto agarose microspheres. Reactive Red 120 is a polycyclic dye with structural similarity to NADP+ , and can be used for purification of nucleotide-dependent enzymes, lipoproteins, lactate dehydrogenase, plasminogen, peptides, hormones, etc. In addition to its use as an affinity filler, the ligand of this product contains aromatic rings and anionic groups, enabling non-affinity purification through electrostatic or hydrophobic interactions.
Aladdin Red Sepharose 6FF is supplied in 20% ethanol, with a gel to preservative solution volume ratio of 1:1. The product specification refers to the actual gel volume.
Parameter | Specification | Matrix | 6% Cross-linked Agarose | Ligand | Reactive Red 120, ~2 μmol/mL | Particle Size Range ① | 45–165 μm | Mean Particle Size | ~90 μm | Dynamic Binding Capacity ② | ~15 mg BSA/mL | Recommended Operating Flow Rate | 60–300 cm/h | Maximum Flow Rate & Pressure ③ | 1500 cm/h, 0.5 MPa | Working pH | 4–8.5 (recommended); 3–12 (long-term stable); 2–14 (short-term stable) | Chemical Stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 6 mol/L guanidine hydrochloride, 70% ethanol | Storage | 20% ethanol, 2–8℃ | Shelf Life | 5 years |
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Notes:
①More than 90% of microspheres by volume are within this range.
② DBC10% tested at 10% breakthrough, 6 min residence time.
③ Maximum tested flow rate at 10 cm bed height.
Instructions for Use
1. Column Packing
The following describes column packing procedures for connection to a chromatography system.
(1)Equilibrate all materials to the temperature of chromatography; degas liquids if possible.
(2)Calculate filler volume: Settled filler volume = Column volume × Compression factor. Settled volume is the stable volume after full sedimentation of the filler in 20% ethanol storage solution. The compression factor of Red Sepharose 6FF is 1.15. Achieve target compression by lowering the top adapter or high-flow packing.
(3)Wash filler: Mix the filler suspension thoroughly, measure the required volume, remove liquid by vacuum filtration, and wash with ~3 column volumes of purified water (repeat 3 times) to remove storage solution.
(4)Prepare packing suspension: Transfer filler to a suitable container, add appropriate packing buffer to make 50–75 v% suspension, and mix well before use.
(5)Pre-packing preparation: Add packing buffer to the bottom of the cleaned column to eliminate air from the lower frit and column base; leave a small volume of packing buffer, tighten the lower end cap, and vertically align the column.
(6)Packing: Slowly pour the well-mixed filler suspension into the column in one portion (use a packing reservoir if needed), running it down the inner wall to avoid bubbles. After adding all filler, fill the packing reservoir with packing buffer, fasten the top cap, and connect the column to the system.
(7)Compaction: Let filler settle naturally (or at low flow rate 50–150 cm/h) until the bed–liquid interface is clear. Remove the packing reservoir, install the top adapter, and lower the adapter to the interface. Continue compaction at high flow rate (see table; keep pressure <0.3 MPa) until the interface is stable; mark the bed height. Stop the pump, open the adapter vent/cap, close the column bottom valve/cap, lower the adapter to the position corresponding to the compression factor below the mark, and tighten. Equilibrate the packed bed at high flow rate.
Packing Condition | Red Sepharose 6FF | Compression Factor | 1.15 | Packing Flow Rate | 600 cm/h |
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2. Column Efficiency Measurement & Evaluation
Column efficiency can be tested after packing and before use to verify quality, using HETP (Height Equivalent to a Theoretical Plate) and asymmetry factor (As).Test with acetone or NaCl solution as sample, according to the table below.
Sample | 1.0% Acetone | 0.8–1.0 M NaCl | Sample Volume | 1.0% of column volume | 1.0% of 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, theoretical plate number (N), and asymmetry factor (As) from UV or conductivity profiles:
HETP = L / N
N = 5.54 × (VR/ Wh)2
As = a / b
Where:L = bed height; VR = retention volume; Wh = peak width at half height;a = first half-width at 10% peak height; b = second half-width at 10% peak height.
General criteria: HETP < 3 × mean particle size (HETP/D₅₀ < 3); As = 0.8–1.5.
3. Equilibration & Loading
Equilibrate the column with Buffer A at operating flow rate before loading, typically 5–10 column volumes until conductivity, pH, and other signals are constant and match Buffer A. Common buffers such as phosphate or Tris-HCl are suitable.
Sample loading can be set as mg target protein per mL filler, usually 50–80% of DBC10%. For this medium (DBC10%~15 mg/mL), load 7.5–12 mg/mL. Alternatively, determine loading by target protein in flow-through.
Centrifuge samples (≥10000 g) and filter (0.22 or 0.45 μm) before loading to prevent clogging.After loading, re-equilibrate with 3–10 column volumes of Buffer A.
4. Elution
Target protein is typically eluted with Buffer A supplemented with 3 M NaCl or 2 M KCl.
5. Regeneration
After purification, regenerate the column by alternating washes with weakly acidic and weakly basic buffers 3–4 cycles, e.g., 0.1 mol/L NaAc + 0.5 mol/L NaCl (pH 4.5) and 0.1 mol/L Tris·HCl + 0.5 mol/L NaCl (pH 8.5). Re-equilibrate with equilibration buffer afterward.
6. Cleaning-in-Place (CIP)
Standard elution and regeneration usually suffice for thorough cleaning. For increased backpressure or cross-contamination prevention, use: 0.1–0.5 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP effectively removes impurities; reverse-flow cleaning is more efficient. After CIP, wash with 3–5 column volumes of pure water to remove cleaning agents.
CIP Procedure | Purpose | 0.1–0.5 mol/L NaOH, ~0.5 h contact time (longer for endotoxin removal); Buffer A, 5–10 CV; Water, 3–5 CV | Remove protein precipitates, hydrophobic proteins, pyrogens, etc. | 30% Isopropanol or 70% ethanol, 5–10 CV; Water, 3–5 CV | Remove tightly bound lipoproteins, liposomes, etc. |
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7. Storage
The medium is supplied in 20% ethanol. After use, store in 20% ethanol with 0.5 M NaCl added. Recommended storage temperature: 2–8℃. Do not freeze.
ZJ26F0636577
危险品化学品经营许可证（带存储）