Blue亲和层析介质是一种用于纯化白蛋白、干扰素、需要核苷酸辅助的酶、α2-巨球蛋白凝血因子等的亲和层析介质。Blue亲和层析介质以高度交联的6%琼脂糖为介质，可耐受较高的流速，具有更高的化学稳定性，适合大规模纯化。
阿拉丁Blue亲和层析介质储存在0.1M K3PO4，20%乙醇，pH8.0 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
| 高度交联的6%琼脂糖微球
|
配基
| 活性蓝3G, 约7µmol/mL 介质
|
| 粒径范围 | 45~165 μm |
载量
| ＞18mg 牛血清白蛋白/mL 介质
|
最大耐压
| 0.3 MPa，3 bar
|
pH 稳定范围
| 4-12 |
储存
| 0.1M K3PO4, 20%乙醇, pH8.0，2~8℃
|
保质期
| 2年 |
使用说明
1、缓冲液的准备
所用水和缓冲液在使用之前建议用0.22μm或0.45μm滤膜过滤。
以人血清白蛋白（HSA）的纯化为例
平衡/洗杂液： 50 mM Na₂HPO₄, 50 mM Sodium Citrate, pH 7.0
洗脱液： 50 mM Na₂HPO₄, 50 mM Sodium Citrate, 1-2 M NaCl, pH 7.0
注：结合液和洗脱液可根据样品性质进行适当改变，原则是低盐上样高盐洗脱。
2、样品准备
样品在上样前建议离心或用0.22μm或0.45μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。
3、Blue亲和层析介质装填
3.1 重力柱的装填
(1) 取合适规格的重力层析柱，装入下垫片，加入适量纯水润洗柱管和垫片，关闭下出口。
(2) 将Blue亲和层析介质混合均匀，用枪头吸取适量浆液加入至重力柱中（介质实际体积占悬液的一半），打开下出口流干保护液。
(3) 加入适量纯水冲洗介质，待柱管中液体重力流干后，关闭下出口。
(4) 装入润洗后的上垫片，确保垫片与填料之前没有空隙，且保持水平。
(5) 装填好的重力柱可以直接加入平衡液进行平衡，暂不使用时则加入保护液，2-8℃保存。
3.2 中压层析柱的装填
Blue亲和层析介质被广泛应用于工业纯化，因此，涉及到各种中压色谱层析柱的填装，下面介绍填装层析柱的方法。
装柱前根据层析柱直径计算柱子底面积，根据所需装柱高度计算所需介质体积，公式如下：
V = 1.15 × π × r² × h
V：所需介质体积，ml
1.15：压缩系数
r：柱管半径，cm
h：装填高度，cm
注意：所取悬液体积应为介质体积的两倍，因为介质体积只占悬液总体积的一半，另一半为保护液。
(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) 将介质悬浮起来，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。
4、样品纯化
4.1 孵育法纯化
(1) 根据纯化的样品量，取适量Blue亲和层析介质加入离心管中，1000rpm离心1min，吸弃上清；也可加入重力柱中，流干保护液。
(2) 向离心管中加入5倍介质体积的平衡液清洗介质，1000rpm离心1min，吸弃上清；如使用重力柱，则直接在重力柱中清洗，直接重力流干平衡液；重复两次以上。
(3) 加入样品，封闭离心管或重力柱管，4℃振荡孵育2-4h或者37℃孵育30min-2h。
(4) 孵育结束后，1000rpm离心1min，吸弃上清，或过滤收集介质，上清保留作为流穿，用于电泳鉴定。
(5) 用5倍介质体积的洗杂液清洗介质，1000rpm离心1min或重力柱管过滤，去除上清（注意不要吸到介质），重复3-5次，中间建议更换新离心管。
(6) 加入3-5倍柱体积的洗脱液进行洗脱，室温孵育5min，1000rpm离心1min或重力柱管收集洗脱液，可重复2-3次。
4.2 重力柱法纯化
(1) 将装填好的Blue亲和层析介质重力柱用5倍柱体积平衡液进行平衡，使填料处于与目的蛋白相同的缓冲液体系下，重复2-3次。
(2) 将样品加到平衡好的重力柱中，样品保留时间至少2min，保证样品和介质充分接触，收集流出液，可以反复上样增加结合效率。
(3) 用10-15倍柱体积的洗杂液进行洗杂，去除非特异性吸附的杂蛋白，收集洗杂液。
(4) 使用5-10倍柱体积的洗脱液洗脱，分段收集，每一个柱体积收集一管，分别检测，既可以保证所有结合的目的蛋白被洗脱，又可以得到高纯度和高浓度的蛋白。
4.3 中压层析柱法纯化Blue亲和层析介质装填好后，可以用各种常规的中低压色谱系统。
(1) 将泵管道中注满去离子水。去掉上塞子，将层析柱连接至色谱系统中，打开下出口，将预装柱接到色谱系统中，并旋紧。
(2) 用3-5倍柱体积的去离子水冲洗出储存缓冲液。
(3) 使用至少5倍柱床体积的平衡液平衡色谱柱。
(4) 利用泵或样品环上样。注：样品的粘度增加使得即使上样体积很少，也会导致层析柱很大的反压。上样量不要超过柱子的结合能力。大量的样品体积也可能造成很大的反压，使得进样器更难使用。
(5) 用洗杂液冲洗柱子，直到紫外吸收达到一个稳定的基线（一般至少10-15个柱体积）。
(6) 使用5-10倍柱体积的洗脱液洗脱，收集洗脱液，即目的蛋白组分。介质洗脱结束后，用平衡液冲洗5-10柱体积，然后用纯水冲洗5-10个柱体积，再用20%乙醇冲洗2个柱体积，置于2-8℃保存。
5、SDS-PAGE 检测 将使用纯化产品得到的样品（包括流出组分、洗杂组分和洗脱组分）以及原始样品使用SDS-PAGE检测纯化效果。
6、填料清洗
Blue亲和层析介质纯化产品可以重复使用而无需再生，但随着非特异性结合的蛋白的增多和蛋白的聚集，往往造成流速和结合载量都下降，这时可按照下面方法对填料进行清洗。
去除一些沉淀或变性物质，建议使用下面的方法
用3-4倍柱体积的0.1M NaOH，然后用3-4倍柱体积的70%乙醇或2M硫氰酸钾溶液清洗，然后立即用5倍柱体积的结合液平衡。
去除一些疏水性吸附造成的非特异性吸附物质
用3-4倍柱体积的70%乙醇或30%的异丙醇或1%Triton™X-100清洗，然后立即用5倍柱体积的结合液平衡。
Blue Agarose Resin is an affinity chromatography medium used for the purification of albumin, interferon, nucleotide-dependent enzymes, α2-macroglobulin, coagulation factors, etc. Blue Affinity Chromatography Medium is based on a highly cross-linked 6% agarose matrix, which can withstand relatively high flow rates, offers higher chemical stability, and is suitable for large-scale purification.
Aladdin Blue Agarose Resin is stored in 0.1 M K₃PO₄, 20% ethanol, pH 8.0, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Specification
| Indicator
|
Matrix
| Highly cross-linked 6% agarose microspheres
|
Ligand
| Cibacron Blue 3G, approx. 7 µmol/mL medium
|
| Particle Size Range | 45 - 165 µm |
Binding Capacity
| >18 mg Bovine Serum Albumin / mL medium
|
| Maximum Pressure Tolerance | 0.3 MPa, 3 bar
|
pH Stability Range
| 4-12 |
Storage
| 0.1 M K₃PO₄, 20% ethanol, pH 8.0, 2~8°C
|
Shelf Life
| 2 years |
Instructions for Use
1. Buffer Preparation
It is recommended to filter all water and buffers using a 0.22 µm or 0.45 µm filter membrane before use.
Using Human Serum Albumin (HSA) purification as an example:
Equilibration/Wash Buffer: 50 mM Na₂HPO₄, 50 mM Sodium Citrate, pH 7.0
Elution Buffer: 50 mM Na₂HPO₄, 50 mM Sodium Citrate, 1-2 M NaCl, pH 7.0
Note: The binding and elution buffers can be adjusted appropriately based on the sample properties. The general principle is low salt concentration for binding and high salt concentration for elution.
2. Sample Preparation
It is recommended to centrifuge the sample or filter it through a 0.22 µm or 0.45 µm filter membrane before loading to reduce impurities, improve purification efficiency, and prevent column clogging.
3. Packing the Blue Affinity Chromatography Medium
3.1 Gravity Column Packing
(1) Take a gravity column of appropriate size, insert the bottom frit, add an appropriate amount of pure water to rinse the column tube and frit, and close the bottom outlet.
(2) Mix the Blue Agarose Resin uniformly. Using a pipette tip, aspirate an appropriate amount of the slurry and add it to the gravity column (the settled medium volume constitutes half of the suspension volume). Open the bottom outlet to drain the storage solution.
(3) Add an appropriate amount of pure water to rinse the medium. After the liquid in the column has drained by gravity, close the bottom outlet.
(4) Insert the rinsed top frit, ensuring no gap between the frit and the medium and that it remains level.
(5) The packed gravity column can be equilibrated directly by adding equilibration buffer. When not in use immediately, add storage solution and store at 2-8°C.
3.2 Medium-Pressure Chromatography Column Packing
Blue Agarose Resin is widely used in industrial purification, thus involving the packing of various medium-pressure chromatography columns. The method for packing a chromatography column is described below.
Before packing, calculate the cross-sectional area of the column based on its diameter, and calculate the required medium volume based on the desired bed height using the formula:
V = 1.15 × π × r² × h
V: Required medium volume (mL)
1.15: Compression factor
r: Column inner radius (cm)
h: Packed bed height (cm)
Note: The volume of suspension taken should be twice the calculated medium volume (V), because the settled medium volume constitutes only half of the total suspension volume; the other half is storage solution.
(1) Rinse the column bottom sieve plate and adaptor with deionized water. Ensure no bubbles are trapped under the bottom sieve plate. Close the column outlet, leaving 1-2 cm of deionized water in the bottom of the column.
(2) Resuspend the medium. Carefully pour the slurry continuously into the column. Pouring the slurry along the column wall using a glass rod can help reduce bubble formation.
(3) If using a reservoir, immediately fill both the column and the reservoir with water. Place the inlet adaptor on the slurry surface and connect it to the pump, avoiding air bubbles in the adaptor or inlet tubing.
(4) Open the column outlet and start the pump at the set flow rate. Initially, allow the buffer to flow through the column slowly, then gradually increase to the final packing flow rate. This avoids hydraulic on the forming bed and prevents uneven packing. If the recommended pressure or flow rate cannot be achieved, using the maximum flow rate of your pump can also yield a good packing result. (Note: In subsequent chromatography runs, do not exceed 75% of the maximum packing flow rate.) When the bed height stabilizes, continue pumping at least 3 bed volumes (BV) of deionized water at the final packing flow rate. Mark the final bed height.
(5) Turn off the pump and close the column outlet.
(6) If a reservoir was used, remove it and position the inlet adaptor within the column tube.
(7) Carefully push the adaptor down to the marked bed height. Allow the packing buffer to enter the adaptor, then tighten the adaptor lock nut.
(8) Connect the packed column to the pump or chromatography system and begin equilibration. Readjust the adaptor if necessary.
4. Sample Purification
4.1 Batch/Binding Procedure
(1) According to the sample volume, transfer an appropriate amount of Blue Affinity Chromatography Medium to a centrifuge tube. Centrifuge at 1000 rpm for 1 min and aspirate the supernatant. Alternatively, add the medium to a gravity column and drain the storage solution.
(2) Add 5 volumes of equilibration buffer (relative to the medium volume) to wash the medium. Centrifuge at 1000 rpm for 1 min and aspirate the supernatant. If using a gravity column, wash directly in the column by draining the equilibration buffer. Repeat this wash step more than twice.
(3) Add the sample. Seal the centrifuge tube or gravity column. Incubate with shaking for 2-4 hours at 4°C or for 30 min - 2 hours at 37°C.
(4) After incubation, centrifuge at 1000 rpm for 1 min and aspirate the supernatant, or filter to collect the medium. Retain the supernatant as the flow-through for SDS-PAGE analysis.
(5) Wash the medium with 5 volumes of wash buffer. Centrifuge at 1000 rpm for 1 min or filter using the gravity column, and remove the supernatant (be careful not to aspirate the medium). Repeat this wash 3-5 times; it is advisable to change to a new centrifuge tube during the process.
(6) Add 3-5 volumes of elution buffer for elution. Incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect the eluate from the gravity column. This elution step can be repeated 2-3 times.
4.2 Gravity Column Procedure
(1) Equilibrate the packed Blue Affinity Chromatography Medium gravity column with 5 column volumes (CV) of equilibration buffer. Repeat 2-3 times to ensure the medium is in the same buffer system as the target protein.
(2) Load the sample onto the equilibrated column. Allow the sample to remain in contact with the medium for at least 2 minutes to ensure sufficient binding. Collect the flow-through. Reloading the flow-through can increase binding efficiency.
(3) Wash the column with 10-15 CV of wash buffer to remove non-specifically bound impurities. Collect the wash fraction.
(4) Elute the bound protein using 5-10 CV of elution buffer. Collect the eluate in fractions (e.g., 1 CV per tube) and analyze them separately. This ensures complete elution of the bound target protein while obtaining high purity and concentration.
4.3 Medium-Pressure Column Procedure
After the Blue Affinity Chromatography Medium is packed, it can be used with conventional medium/low-pressure chromatography systems.
(1) Prime the pump tubing with deionized water. Remove the top cap, connect the column to the chromatography system, open the bottom outlet, and secure the connection.
(2) Flush out the storage buffer with 3-5 CV of deionized water.
(3) Equilibrate the column with at least 5 CV of equilibration buffer.
(4) Load the sample using a pump or a sample loop. Note: Increased sample viscosity can lead to high backpressure even with small sample volumes. Do not exceed the binding capacity of the column. Large sample volumes can also cause high backpressure, making injector use more difficult.
(5) Wash the column with wash buffer until the UV absorbance reaches a stable baseline (typically at least 10-15 CV).
(6) Elute with 5-10 CV of elution buffer. Collect the eluate, which contains the target protein. After elution, wash the column with 5-10 CV of equilibration buffer, followed by 5-10 CV of deionized water. Finally, wash with 2 CV of 20% ethanol for storage. Store at 2-8°C.
5. SDS-PAGE Analysis
Analyze the samples obtained from the purification process (including flow-through, wash, and elution fractions) as well as the original crude sample using SDS-PAGE to evaluate the purification effectiveness.
6. Medium Cleaning
Blue Affinity Chromatography Medium can be reused multiple times without mandatory regeneration. However, the accumulation of non-specifically bound proteins and protein aggregates can lead to decreased flow rates and binding capacity. The following cleaning procedures can be performed when necessary.
To remove precipitated or denatured substances, the following method is recommended:
Wash with 3-4 CV of 0.1 M NaOH, followed by 3-4 CV of 70% ethanol or 2 M potassium thiocyanate solution. Then, immediately equilibrate with 5 CV of binding buffer.
To remove non-specifically bound substances caused by hydrophobic adsorption:
Wash with 3-4 CV of 70% ethanol, or 30% isopropanol, or 1% Triton™ X-100. Then, immediately equilibrate with 5 CV of binding buffer.
ZJ26F0738002
危险品化学品经营许可证（带存储）