储存缓冲液：20% (v/v) Ethanol
本产品是一款应用于内毒素去除的琼脂糖凝胶填料，配基为多粘菌素B（Polymyxin B），能保持较高的分辨率和重力流速。
多粘菌素 B 能够特异性地结合革兰氏阴性菌的脂多糖（LPS），在温和条件下可高效结合内毒素，去除率高且样品回收率高。经内毒素去除介质纯化后，蛋白溶液中的内毒素含量可降低至0.1~0.25 EU/mL。填料可耐受不同 pH、离子强度的缓冲体系，可应用于生物制药、蛋白纯化以及其他生物制品的内毒素去除工艺。
阿拉丁内毒素去除介质储存在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%高度交联琼脂糖
| 配基
| 多粘菌素B，~2 mg/mL
| 粒径范围 ①
| 30~70 μm
| 平均粒径
| ~50 μm
| | 内毒素结合能力④ | ~2000000 EU/mL
| 推荐工作流速 ②
| 30~150 cm/h
| 最大流速与压力 ③
| 300 cm/h，0.3 MPa
| 使用pH
| 3~10（推荐的工作pH），3~12（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、300 mM咪唑、10 mM DTT等。
| 储存
| 20%乙醇，2~8℃
| 保质期
| 3年 |
|
注：
① 90%体积以上的微球在此粒径范围；
② 推荐流速是指该流速充分的接触时间；
③ 10cm柱高下的最大测试流速。
④ 内毒素结合能力 (~2000000 EU/mL) 的测定需满足：原始样本内毒素浓度达到一定的检测阈值；且结合效率与内毒素含量呈正相关，相关参考数据见下表。
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。内毒素去除介质的压缩比为1.20。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
| 内毒素去除介质
| 压缩比
| 1.20
| 装柱流速
| ~150 cm/h |
|
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 |
|
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 × (Vʀ / Wₕ)²
As = a / b
其中：L为柱高； Vʀ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、再生
通常在使用首次使用前和每次使用后对填料进行再生，以确保填料的内毒素含量尽可能低。
常用的再生溶液为1% Triton X-114（使用温度：2~8℃）或者1% DOC（脱氧胆酸钠）。可用5~10个柱体积的溶液对色谱柱进行再生，再生后用5~10个柱体积无热源的缓冲液再平衡色谱柱。
4、平衡与上样
实验中使用的水、缓冲液和耗材均需经过无热原处理，以防止在操作过程中引入内毒素。
缓冲液与产物体系尽可能保持一致，例如20 mM PB，150 mM NaCl，pH 7.0~7.4是较为常用的缓冲体系。建议pH的范围为7~8，NaCl的浓度范围为150 mM～500 mM，以降低非特异性吸附和蛋白与内毒素之间的静电作用。
在上样前，需用缓冲液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，以电导、pH等检测信号参数不变且与缓冲液A对应为准。
在足够的接触时间条件下，上样体积与填料体积比通常在1~100，更大的上样体积有利于提高回收率。上样后收集流穿中的目标蛋白。
可根据实际需求再上样1~3次。
5、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%乙醇保存。保存温度在2~8℃为宜，不可冻存。
This product is an agarose-based chromatography medium designed for endotoxin removal, with Polymyxin B as the ligand. It maintains high resolution and gravity flow rates.
Polymyxin B specifically binds to lipopolysaccharides (LPS) from Gram-negative bacteria, enabling efficient endotoxin removal under mild conditions with high recovery rates. After purification with this endotoxin removal resin, endotoxin levels in protein solutions can be reduced to 0.1–0.25 EU/mL. The resin is compatible with buffers of various pH levels and ionic strengths, making it suitable for endotoxin removal in biopharmaceuticals, protein purification, and other biological products.
Aladdin Endotoxin Removal 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
| Value / Description
| Matrix
| 4% highly cross-linked agarose
| Ligand
| Polymyxin B, ~2 mg/mL
| Particle Size Range ①
| 30~70 μm
| Average Particle Size
| ~50 μm
| | Endotoxin Binding Capacity④ | ~2000000 EU/mL
| Recommended Operating Flow Rate ②
| 30~150 cm/h
| Maximum Flow Rate & Pressure ③
| 300 cm/h, 0.3 MPa
| Operating pH Range
| 3–10 (recommended), 3–12 (short-term)
| Chemical Stability
| Stable in common aqueous buffers, 300 mM imidazole, 10 mM DTT, etc.
| Storage Conditions
| 20% ethanol, 2–8°C
| Shelf Life
| 3 years |
|
Notes:
① Over 90% of the beads fall within this size range.
② This flow rate range ensures sufficient contact time for effective binding.
③ Maximum tested flow rate at 10 cm column height.
④The determination of endotoxin-binding capacity (~2000000 EU/mL) requires that: The original sample's endotoxin concentration reaches a certain detection threshold; The binding efficiency is positively correlated with the endotoxin content. Refer to the table below for relevant reference data.
Protocol
1. Column Packing
The following procedure describes column packing when connected to a chromatography system:
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (1.20 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed). After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 50–150 cm/h). Apply a high flow rate (~150 cm/h; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height, stop the pump, and lower the adapter to the position corresponding to the compression factor (1.20). Tighten the adapter and equilibrate the column at a high flow rate.
Packing Condition
| Endotoxin Removal Resin
| Compression Factor
| 1.20
| Packing Flow Rate
| ~150 cm/h |
|
2. Column Efficiency Testing
After packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
| Sample Volume
| 1.0% CV
| 1.0% CV
| Mobile Phase
| Pure Water
| 0.4 M NaCl
| Flow Rate
| 30 cm/h
| 30 cm/h
| Detector
| UV-280 nm
| Conductivity |
|
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Regeneration
Regenerate the resin before first use and after each use to minimize endotoxin levels:
Use 1% Triton X-114 (at 2–8°C) or 1% DOC (sodium deoxycholate).
Wash with 5–10 CV of regeneration solution, followed by 5–10 CV of endotoxin-free buffer.
4. Equilibration and Loading
Use endotoxin-free water, buffers, and consumables to avoid contamination.
Recommended Buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.0–7.4. Adjust pH to 7–8 and NaCl concentration to 150–500 mM to reduce non-specific adsorption and electrostatic interactions.
Equilibrate the column with 5–10 CV of buffer at the operating flow rate until baseline (conductivity/pH) stabilizes.
Load sample at a resin-to-sample volume ratio of 1:1 to 1:100. Larger sample volumes may improve recovery. Collect the flow-through containing the target protein.
Repeat loading 1–3 times if needed.
5. Storage
Store in 20% ethanol at 2–8°C. Do not freeze.
危险品化学品经营许可证（带存储）