葡聚糖G-25层析介质是一类以葡聚糖为基质的凝胶过滤层析（gel filtration chromatography）介质，其工作原理主要是利用具有网状结构的葡聚糖凝胶的分子筛作用，根据被分离物质的分子大小不同来进行分离。
葡聚糖凝胶是一种具有三维网状结构的高分子化合物，它是由葡聚糖加入交联剂通过醚键互相交联聚合而成的，交联度越大，网孔结构就越紧密，吸水后的体积膨胀就越小，能允许进入凝胶内部的分子的分子量也就越小，反之亦然。层析时，大于凝胶孔径的大分子，被阻于凝胶相外，沿着凝胶颗粒之间的间隙走，下移速度最快，故最先洗脱下来，中分子物质部分进入凝胶内部，洗脱速度为其次，而小分子物质因全部进入凝胶，受到阻力最大，故最后被洗脱下来，如此物质得到分离。
型号G表明每克干凝胶吸水量，例如G-25表明每克干凝胶吸水2.5克，这表征了凝胶所能膨胀的倍数，亦间接表征了凝胶孔径的大小。
| 名称 | 葡聚糖G-25层析介质（中） |
| 湿球平均粒径D50（μm） | 120-140 |
| 分离范围 （葡聚糖） | 1×10² - 5×10³
|
| 分离范围 （球蛋白） | 1×10³ - 5×10³
|
| 溶胀因子 （ml/g） | 4-6 |
| pH 稳定范围 | 2-13 |
| 最大流速 （cm/h） | 150 |
葡聚糖G-25系列层析介质常用于蛋白溶液的脱盐，缓冲液交换和不同分子量蛋白的分离等，根据不同的用途其上样量也不可一概而论，样品溶液的缓冲液置换时最高可上样30%柱体积。
装柱说明
1、凝胶的预处理
葡聚糖G-25层析介质为干粉，初次使用前必须进行预处理。溶胀过程中严禁使用磁力搅拌，以免使微球破碎。
加入足够的水或缓冲液进行溶胀，室温3h以上或水浴90℃ 1h。如果室温溶胀则需要进行真空脱气，然后将溶胀好的微球按照3：1（v/v）加入水或缓冲液搅拌使其形成均匀的75%胶悬液。如果是重复使用的可以将20%乙醇倾去，加水或缓冲液搅拌均匀成75%的胶悬液。
2、层析柱的装填（使用储液器装填）
(1) 用去离子水冲洗层析柱底筛板与接头，确保柱底筛板上无气泡，关闭柱底出口，并在柱底部留出1-2cm的去离子水。
(2) 将填料悬浮起来，小心的将浆液连续地倒入层析柱中。用玻璃棒沿着柱壁倒入浆液可减少气泡的产生。
(3) 如果使用储液器，应立即在层析柱和储液器中加满水，将进样分配器放置于浆液表面，连接至泵上，避免在分配器或进样管中产生气泡。
(4) 打开层析柱底部出口，开启泵，使其在设定的流速下进行。最初应让缓冲液缓慢流过层析柱，然后缓慢增加至最终流速，这样可避免液压对所形成柱床的冲击，也可以避免柱床形成的不均匀。如果达不到推荐的压力或流速，可以用你所使用泵的最大流速，这样也可以得到一个很好的装填效果。（注意：在随后的色谱程序中，不要超过最大装柱流速的75%）当柱床高度稳定后，在最后的装柱流速下至少再上3倍柱床体积的去离子水。标上柱床高度。
(5) 关闭泵，关闭层析柱出口。
(6) 如果使用储液器，去除储液器，将分配器置于层析柱中。
(7) 将分配器推向柱子至标记的柱床高度处。允许装柱液进入分配器，锁紧分配器接头。
(8) 将装填好的层析柱连接至泵或色谱系统中，开始平衡。如果需要可以重新调整分配器。
纯化流程
缓冲液：期望置换到的缓冲液
(1) 柱平衡：用缓冲液平衡脱盐柱5-10CV。
(2) 样品：建议上样量小于10%柱体积，脱盐时最大样品量可达30%柱体积。为了延长填料的使用寿命，所用的样品推荐使用0.45μm的滤膜过滤。
(3) 洗脱：样品洗脱过程中使用的流速不要超过推荐的最大流速。凝胶过滤是一种非吸附性色谱技术，所有的样品物质都应在一个柱体积之内洗脱出来。完成一个操作后，如仍用同一种缓冲液，色谱柱不需要再平衡。
清洗及保存
在位清洗：为除去变性蛋白或脂肪，有时需要对脱盐柱进行原位清洗。常用的清洗液为0.1-0.2M NaOH或非离子型去垢剂。
(1) 加入一倍的柱体积0.1-0.2M NaOH，依靠重力或者低流速清洗介质。
(2) 用足够量的去离子水，冲洗介质，直至pH值至中性。
(3) 用至少三倍柱体积灭菌水冲洗介质，然后做好色谱柱的密封，置于2-8℃保存。
注：不建议介质多次重复再生和使用。
Dextran G-25 Chromatography Media is a type of gel filtration chromatography medium based on dextran. Its working principle primarily relies on the molecular sieving effect of the dextran gel's network structure, separating substances based on differences in their molecular sizes.
Dextran gel is a high-molecular-weight compound with a three-dimensional network structure. It is formed by cross-linking dextran with cross-linking agents through ether bonds. A higher degree of cross-linking results in a tighter pore structure, less volume expansion upon water absorption, and a smaller molecular weight cutoff for molecules entering the gel interior, and vice versa.
During chromatography, large molecules larger than the gel pores are excluded from the gel phase and move fastest through the inter-particle spaces, eluting first. Medium-sized molecules partially enter the gel pores and elute next. Small molecules enter the gel pores entirely, experiencing the greatest resistance, and thus elute last. In this way, substances are separated.
The "G" model number indicates the water regain per gram of dry gel. For example, G-25 indicates 2.5 grams of water per gram of dry gel. This reflects the gel's swelling capacity and indirectly characterizes the pore size.
| Name | Dextran G-25 (Medium) |
| Wet bead average particle size D50 (μm) | 120-140 |
| Separation range (Dextran) | 1×10² - 5×10³
|
| Separation range (Globular proteins) | 1×10³ - 5×10³
|
| Swelling factor (ml/g dry medium) | 4-6 |
| pH stability range | 2-13 |
| Maximum flow rate (cm/h) | 150 |
The Dextran G-25 series chromatography media are commonly used for desalting protein solutions, buffer exchange, and separation of proteins with different molecular weights. The loading volume varies depending on the application and cannot be generalized. For buffer exchange of sample solutions, up to 30% of the column volume can be loaded.
Column Packing Instructions
1. Gel Pre-treatment
Dextran G-25 Chromatography Media is supplied as a dry powder and must be pre-treated before first use. Do not use magnetic stirring during swelling to avoid breaking the beads.
Add sufficient water or buffer for swelling: either at room temperature for over 3 hours or in a water bath at 90°C for 1 hour. If swelling at room temperature, degas under vacuum afterwards. Then, add water or buffer to the swollen beads at a 3:1 (v/v) ratio and stir to form a homogeneous 75% gel suspension. For reused media, decant the 20% ethanol storage solution, add water or buffer, and stir into a 75% gel suspension.
2. Packing the Chromatography Column (Using a Reservoir)
(1) Rinse the column bottom frit and adaptor with deionized water to ensure no air bubbles are trapped. Close the column outlet and leave 1-2 cm of deionized water at the column bottom.
(2) Resuspend the prepared gel slurry and carefully pour it continuously into the column. Pouring along the column wall using a glass rod can reduce bubble formation.
(3) If using a reservoir, immediately fill both the column and reservoir with water. Place the packing adapter on the slurry surface and connect it to the pump, avoiding air bubbles in the adapter or tubing.
(4) Open the column outlet and start the pump at the set flow rate. Initially, allow the buffer to flow slowly through the column, then gradually increase to the final packing flow rate. This avoids hydraulic shock to the forming bed and prevents uneven packing. If the recommended pressure/flow cannot be reached, use the pump's maximum flow rate; this can also yield good packing results. (Note: In subsequent chromatography runs, do not exceed 75% of the maximum packing flow rate.) Once the bed height stabilizes, run at least 3 column volumes (CV) of deionized water at the final packing flow rate. Mark the final bed height.
(5) Stop the pump and close the column outlet.
(6) If a reservoir was used, remove it and place the top flow adapter into the column.
(7) Lower the adapter until it touches the marked bed height. Allow packing liquid to enter the adapter, then tighten the adapter lock nut.
(8) Connect the packed column to the pump or chromatography system and begin equilibration. Readjust the adapter if necessary.
Purification Protocol
Buffer: The target buffer for exchange.
(1) Column Equilibration: Equilibrate the desalting column with 5-10 CV of buffer.
(2) Sample: The recommended loading volume is less than 10% of the column volume. For desalting, the maximum sample volume can be up to 30% CV. To prolong media life, it is recommended to filter the sample through a 0.45 μm membrane.
(3) Elution: Do not exceed the recommended maximum flow rate during sample elution. Gel filtration is a non-adsorptive technique; all sample material should elute within one column volume. After a run, if the same buffer will be used next, the column does not require re-equilibration.
Cleaning and Storage
Cleaning-in-Place (CIP): To remove denatured proteins or lipids, in-place cleaning of the desalting column is sometimes necessary. Common cleaning solutions are 0.1-0.2 M NaOH or non-ionic detergents.
(1) Introduce one column volume of 0.1-0.2 M NaOH and let it flow through the media by gravity or at a low flow rate.
(2) Rinse the media with a sufficient amount of deionized water until the effluent pH is neutral.
(3) Flush the media with at least 3 CV of sterile water. Then, seal the column properly and store at 2-8°C.
Note: Repeated regeneration and reuse of the media is not recommended.
ZJ26F0433892
危险品化学品经营许可证（带存储）