本产品是一种环氧活化的琼脂糖微球，可以直接用于含氨基、巯基和羟基的蛋白和样品的耦联。预活化介质可以根据需要制备成特殊的亲和介质，快速有效地从复杂体系中一步纯化相应的物质。
阿拉丁环氧活化亲和层析介质4FF储存在100% 1,4-二氧六环中，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4%琼脂糖微球
|
活性基团密度
| 30-40 μmol/mL 介质
|
粒径范围
| 45~165 μm
|
最大压力
| 0.3 MPa，3 bar
|
储存缓冲液
| 100% 1,4-二氧六环
|
储存温度
| 4-30℃ |
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。
偶联液：0.1 M Na₂CO₃, pH 8.5-10.0
封闭液：1M 乙醇胺，pH8.0
清洗液1：0.1M 乙酸-乙酸钠，0.5M NaCl，pH3.0
清洗液2：0.1M Tris-HCl，0.5M NaCl，pH8.0
保护液：含20%乙醇的1×PBS
2、样品准备
样品用偶联液溶解或透析，浓度约5-10mg/mL。
3、样品偶联
下面以偶联抗体纯化抗原为例，介绍偶联及后续纯化步骤。
3.1 取适量的环氧活化亲和层析介质4FF，去除保护液，切勿抽干，去离子水清洗三次，用偶联液清洗一次。
3.2 将溶解好的样品中溶解后转入清洗好的环氧活化亲和层析介质4FF中，填料：样品溶液体积比约为1：1-2（V/V）。
3.3 25-40℃振荡混合反应24h。
注：确保填料悬浮起来，否则会大大影响偶联效率。
3.4 反应完后收集偶联样品，以便检测偶联效率。偶联液清洗一遍。
3.5 加入等体积的封闭液，37℃振荡混合反应1h。
3.6 将上述反应体系取出，流干其中的溶液，用3倍柱体积的去离子水清洗填料，清洗液1、去离子水、清洗液2和去离子水重复冲洗2次， 然后保存在等体积的保护液中，于2-8℃保存。
4、层析柱的装填
4.1 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) 将偶联好的填料混合均匀，用枪头吸取适量浆液加入至重力柱中，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。
4.2 中压层析柱的装填
偶联好的填料也可用于大体积样品的纯化，因此，涉及到各种中压色谱层析柱的填装，下面介绍使填装层析柱的方法。
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。
5、样品纯化
5.1 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。
平衡/ 洗杂液：0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0
洗脱液：0.1M 甘氨酸，pH3.0
中和液：1M Tris-HCl，pH8.5
5.2 孵育法纯化
(1) 根据纯化的样品量，取适量偶联好的填料加入层析柱中，重力流干保护液。
(2) 加入5倍介质体积的平衡液清洗介质，重力流干。
(3) 加入样品，封闭柱管两端，4℃振荡孵育2-4h或者37℃孵育30min-2h。
(4) 孵育结束后，离心或过滤收集介质，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，离心或过滤去除上清，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，孵育10-15min，离心或过滤收集洗脱液，可重复2-3次。洗脱组分需要立即调成中性，一般建议使用洗脱组分体积1/10的中和液进行中和。
5.3 重力柱法纯化
(1) 将装填好的重力柱用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。
(2) 将样品加到平衡好的重力柱中，收集流出液，可以反复上样增加结合效率。
(3) 用10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倍柱体积的洗脱液洗脱，分管收集。洗脱组分需要立即调成中性，一般建议使用洗脱组分体积1/10的中和液进行中和。
5.4 中压层析柱法纯化
中压层析柱装填好后，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。洗脱组分需要立即调成中性，一般建议使用洗脱组分体积1/10的中和液进行中和。
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。
6、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。
This product is an epoxy-activated agarose microsphere medium that can be directly used for coupling proteins and samples containing amino, thiol, and hydroxyl groups. The pre-activated medium can be customized to prepare specific affinity media for the rapid and efficient one-step purification of target substances from complex systems.
Aladdin Epoxy-activated Agarose Resin 4FF is stored in 100% 1,4-dioxane, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Specification
|
Matrix
| Highly Cross-linked 4% Agarose Microspheres
|
| Active Group Density | 30-40 μmol/mL medium
|
Particle Size Range
| 45~165 μm
|
Max Pressure
| 0.3 MPa, 3 bar
|
Storage Buffer
| 100% 1,4-Dioxane
|
Storage Temperature
| 4-30℃ |
Shelf Life
| 2 years |
Instructions for Use
1. Buffer Preparation
It is recommended to filter all water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Coupling Buffer: 0.1 M Na₂CO₃, pH 8.5-10.0
Blocking Buffer: 1 M Ethanolamine, pH 8.0
Wash Buffer 1: 0.1 M Sodium Acetate-Acetic Acid, 0.5 M NaCl, pH 3.0
Wash Buffer 2: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0
Storage Buffer: 1× PBS containing 20% Ethanol
2. Sample Preparation
Dissolve or dialyze the sample in Coupling Buffer to a concentration of approximately 5-10 mg/mL.
3. Sample Coupling
The following procedure uses antibody coupling for antigen purification as an example to describe the coupling and subsequent purification steps.
3.1 Take an appropriate amount of Epoxy-activated Agarose Resin 4FF. Remove the storage solution. Do not let the medium dry out. Wash three times with deionized water and once with Coupling Buffer.
3.2 Transfer the dissolved sample to the washed medium. The recommended medium-to-sample solution volume ratio is approximately 1:1-2 (V/V).
3.3 Mix and react with shaking at 25-40°C for 24 hours.
Note: Ensure the medium is suspended, otherwise the coupling efficiency will be significantly affected.
3.4 After the reaction, collect the coupling solution to check coupling efficiency. Wash the medium once with Coupling Buffer.
3.5 Add an equal volume of Blocking Buffer. Mix and react with shaking at 37°C for 1 hour.
3.6 Drain the solution from the reaction system. Wash the medium with 3 column volumes (CV) of deionized water. Rinse alternately with Wash Buffer 1, deionized water, Wash Buffer 2, and deionized water, repeating this cycle twice. Finally, store the medium in an equal volume of Storage Buffer at 2-8°C.
4. Column Packing
4.1 Gravity Column Packing
(1) Take a gravity column of appropriate size. Insert the bottom frit. Add some purified water to wet the column tube and frit. Close the bottom outlet.
(2) Resuspend the coupled medium. Use a pipette tip to aspirate and transfer an appropriate amount of the slurry into the gravity column. Open the bottom outlet to drain the storage solution.
(3) Add purified water to wash the medium. After the liquid has drained by gravity, close the bottom outlet.
(4) Insert the pre-wetted top frit, ensuring no gaps between the frit and the medium and that it is level.
(5) The packed gravity column can be directly equilibrated with equilibration buffer.
4.2 Medium-Pressure Chromatography Column Packing
Coupled medium can also be used for large-volume sample purification, requiring packing into medium-pressure columns. The method is described below:
Before packing, calculate the column base area based on its diameter and 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 radius (cm)
h: Packing height (cm)
Note: The slurry volume taken should be twice the medium volume, as the medium only occupies half the total slurry volume; the other half is storage solution.
(1) Flush the column bottom screen and adaptor with deionized water to ensure no air bubbles are trapped. Close the bottom outlet, leaving 1-2 cm of deionized water in the column bottom.
(2) Resuspend the medium and carefully pour the slurry continuously into the column. Pouring along the column wall with a glass rod can reduce bubble formation.
(3) If using a reservoir, immediately fill both the column and 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 bottom outlet and start the pump at the set flow rate. Initially, allow buffer to flow slowly through the column, then gradually increase to the final flow rate. This avoids disturbing the formed bed and ensures uniform packing. If the recommended pressure/flow cannot be reached, use the pump's maximum flow rate for acceptable results. (Note: Do not exceed 75% of the maximum packing flow rate in subsequent chromatographic runs.) After the bed height stabilizes, continue pumping at least 3 CV of deionized water at the final packing flow rate. Mark the bed height.
(5) Stop the pump and close the column outlet.
(6) If a reservoir was used, remove it and place the adaptor inside the column.
(7) Lower the adaptor down to the marked bed height. Allow packing buffer to enter the adaptor and tighten the adaptor fitting.
(8) Connect the packed column to the pump or chromatography system and begin equilibration. Readjust the adaptor if necessary.
5. Sample Purification
5.1 Buffer Preparation
It is recommended to filter all water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Equilibration/Wash Buffer: 0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0
Elution Buffer: 0.1 M Glycine, pH 3.0
Neutralization Buffer: 1 M Tris-HCl, pH 8.5
5.2 Batch Purification (Incubation)
(1) Based on the sample volume, add an appropriate amount of coupled medium to a column. Drain the storage solution by gravity.
(2) Wash the medium with 5 CV of Equilibration Buffer. Drain by gravity.
(3) Add the sample. Seal both ends of the column and incubate with shaking at 4°C for 2-4 hours or at 37°C for 30 minutes - 2 hours.
(4) After incubation, collect the medium by centrifugation or filtration. Retain the supernatant as the flow-through for SDS-PAGE analysis.
(5) Wash the medium with 5 CV of Wash Buffer. Remove the supernatant by centrifugation or filtration. Repeat this wash step 3-5 times. It is recommended to use a new tube during the process.
(6) Add 3-5 CV of Elution Buffer for elution. Incubate for 10-15 minutes. Collect the eluate by centrifugation or filtration. This elution step can be repeated 2-3 times. The eluted fractions must be neutralized immediately. It is generally recommended to add a volume of Neutralization Buffer equal to 1/10th of the elution fraction volume.
5.3 Gravity Column Purification
(1) Equilibrate the packed gravity column with 5 CV of Equilibration Buffer to bring the medium into the same buffer system as the target protein.
(2) Apply the sample to the equilibrated column. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10 CV of Wash Buffer to remove non-specifically adsorbed impurities. Collect the wash fractions.
(4) Elute with 5 CV of Elution Buffer. Collect fractions separately. Neutralize the eluted fractions immediately by adding a volume of Neutralization Buffer equal to 1/10th of the elution fraction volume.
5.4 Medium-Pressure Column Purification
Once packed, the medium-pressure column 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, attach the pre-packed column to the system, and tighten.
(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 sample loop. Note: Increased sample viscosity can cause 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 the injector harder to use.
(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. Neutralize the eluted fractions immediately by adding a volume of Neutralization Buffer equal to 1/10th of the elution fraction volume.
After elution, wash the medium with 5-10 CV of Equilibration Buffer, followed by 5-10 CV of purified water, and finally 2 CV of 20% ethanol. Store at 2-8°C.
6. SDS-PAGE Analysis
Analyze the samples obtained from the purification process (including flow-through, wash, and elution fractions) as well as the original sample using SDS-PAGE to evaluate the purification efficiency.
ZJ25F1028970
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