Co-NTA 亲和层析介质 6FF可以从各种表达系统（如大肠杆菌、酵母、昆虫细胞和哺乳动物细胞）中一步纯化His标签蛋白。该产品是以高度交联的6%琼脂糖凝胶为基质，配体以四配位键整合钴，因其耐压的基质，可以耐受最高0.3 MPa的压力，更适合用于工业大规模蛋白的纯化。Co-NTA 亲和层析介质 6FF还可以耐受一定范围的变性剂和其他的添加剂（见表2）。 Co-NTA 亲和层析介质 6FF 另外一个特点是，相比 Ni-NTA 亲和层析介质 6FF 对His标签蛋白具有更高的选择性。
阿拉丁 Co-NTA 亲和层析介质 6FF储存在20%乙醇的1×PBS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。
表1：Co-NTA 亲和层析介质 6FF产品性能
| 项目 | 性能 |
|---|
| 基质 | 高度交联的6%琼脂糖凝胶 | | 载量 | >20 mg 6×His-tagged protein/ml 介质 | | 微球粒径 | 45–165 μm | | 最大压力 | 0.3 MPa, 3 bar | | 储存缓冲液 | 含20%乙醇的1×PBS | | 储存温度 | 2–8℃ |
|
表2：Co-NTA 亲和层析介质 6FF试剂耐受情况
| 试剂种类 | 浓度/示例 |
|---|
| 还原剂 | 10 mM β-mercaptoethanol① | | 变性剂 | 8 M urea, 6 M Gua-HCl | | 去污剂 | 1% Triton X-100 (nonionic), 1% NP-40 (nonionic), 1% CHAPS (zwitterionic), SDS, sarcosyl | | 其他类 | 500 mM imidazolee②, 30% ethanols③, 20% glycerol, 500 mM KCl, 1 M NaCl |
|
注
①不要将介质保存在含有β-Mercaptoethanol的缓冲液中。
②过高浓度的咪唑有可能会降低蛋白回收率。
③乙醇有可能造成蛋白沉淀，降低蛋白回收率和堵柱子。
避免使用下列试剂：
DTT（dithiothreitol），DTE（dithioerythritol）和TCEP（tris(2-carboxyethyl)phosphine）会降低介质的蛋白载量。EDTA（ethylenediaminetetraacetic acid）、EGTA（ethylene glycol-bis(β-amino-ethyl ether)）会将钴离子从介质上剥落。
1. 纯化流程
1.1 缓冲液的准备
可使用下列推荐缓冲液，也可根据自己的使用习惯配置不同的缓冲液体系，基本原理就是低咪唑上样，高咪唑洗脱。推荐使用中性偏碱缓冲液（pH 7–8），如磷酸盐缓冲液。可加入0.3–0.5 M氯化钠减少非特异性吸附。在蛋白结合力弱的条件下避免使用Tris-HCl。应避免使用螯合剂EDTA和柠檬酸盐缓冲液。缓冲液在使用前最好用0.22 μm或者0.45 μm滤膜过滤。因为Co-NTA 亲和层析介质 6FF可以用于可溶蛋白和包涵体蛋白的纯化，两种方法所需缓冲液不同。具体配置方法见表3和表4。
表3. 可溶性组氨酸标签蛋白纯化所需缓冲液及配方
| 缓冲液名称 | 体积 | 配方 |
|---|
| Lysis Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 使用NaOH溶液调节pH至8.0，使用0.22或0.45 μm滤膜过滤。 | | Wash Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 5 mM imidazole (0.34 g imidazole), 使用NaOH溶液调节pH至8.0，过滤。 | | Elution Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole), 使用NaOH溶液调节pH至8.0，过滤。 |
|
表4. 包涵体组氨酸标签蛋白纯化所需缓冲液及配方（咪唑洗脱方式）
| 缓冲液名称 | 体积 | 配方 |
|---|
| Lysis Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 使用NaOH溶液调节pH至8.0，过滤。 | | Wash Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole), 调节pH至8.0，过滤。 | | Elution Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole), 调节pH至8.0，过滤。 |
|
1.2 样品准备
1.2.1 细菌或酵母表达的蛋白
① 挑取单菌落到培养基中，根据载体使用说明，加入相应浓度的诱导剂诱导相应的时间。
② 表达结束后，将培养液转移到离心杯中，7,000 rpm (7,500×g)，离心15 min收集菌体，然后按照菌体 : Lysis Buffer = 1 : 10 (W/V)加入Lysis Buffer，加入终浓度为1 mM的PMSF。加入溶菌酶（工作浓度为0.2–0.4 mg/ml，如果表达的宿主细胞内含pLysS或pLysE，可以不加溶菌酶），同时也可加入其他蛋白酶抑制剂，但不能影响目的蛋白与介质的结合。
③ 将菌体沉淀悬浮起来，（如果菌液浓度高，也可考虑加入10 μg/ml RNase A和5 μg/ml DNase I），混匀，放置于冰上，然后冰上超声破碎细胞，至菌液基本保持澄清。
④ 将澄清的破碎液转移至离心管中，10,000 rpm (15,000×g)，4℃离心20–30 min。取上清，置于冰上备用或-20℃保存。
1.2.2 酵母、昆虫和哺乳细胞分泌表达可溶性蛋白
① 将细胞培养液转移至离心杯，5,000 rpm (3,800×g)，离心10 min，收集菌体得上清，如上清中不含EDTA、组氨酸和还原剂等物质，即可直接加入柱子使用；如含有EDTA、组氨酸和还原剂等物质，需用Lysis Buffer透析才能加入柱子。
② 对于大量体积的上清，需加入硫酸铵沉淀浓缩后，蛋白还需用Lysis Buffer透析后才能加入柱子。
1.2.3 包涵体蛋白纯化（变性条件）
① 将培养液转移到离心杯中，7,000 rpm (7,500×g)，离心15 min收集菌体，去掉上清。
② 按照菌体 : Lysis Buffer（不含8 M尿素）= 1 : 10 (W/V)将菌体悬浮起来混匀，冰浴超声破碎。
③ 将破碎液转移至离心管中，10,000 rpm (15,000×g)，4℃离心20–30 min。去掉上清，步骤2和3可以重复一次。
④ 按照菌体 : Lysis Buffer（含8 M尿素）= 1 : 10 (W/V)将包涵体悬浮起来。
⑤ 变性条件下进行His标签蛋白纯化，具体缓冲液配方见表4。
1.3 Co-NTA 亲和层析介质 6FF的装填
1.3.1 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Co-NTA 亲和层析介质 6FF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，确保垫片与填料之间没有空隙，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。
1.3.2 中压层析柱的装填
Co-NTA 亲和层析介质 6FF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：所取悬液体积应为介质体积的两倍，因为介质体积只占总悬液体积的一半，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1–2 cm的去离子水。
② 将介质悬浮起来，小心地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的柱床的冲击，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。
1.4 样品纯化流程
1.4.1 孵育法纯化
① 根据纯化的样品量，取适量Co-NTA 亲和层析介质 6FF加入离心管中，1000 rpm离心1 min，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Lysis Buffer清洗介质，1000 rpm离心1 min，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Lysis Buffer；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2–4 h或者37℃孵育30 min–2 h。
④ 孵育结束后，1000 rpm离心1 min，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Wash Buffer清洗介质，1000 rpm离心1 min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。必要时可以调整咪唑的浓度进行洗涤。
⑥ 加入3–5倍柱体积的Elution Buffer进行洗脱，室温孵育10–15 min，1000 rpm离心1 min或重力柱管收集洗脱液，可重复2–3次。
1.4.2 重力柱法纯化
① 将装填好的Co-NTA 亲和层析介质 6FF重力柱用5倍柱体积Lysis Buffer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2 min，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Wash Buffer进行洗涤，去除非特异性吸附的杂蛋白，收集洗涤液。必要时可以调整咪唑的浓度进行洗涤。
④ 使用5–10倍柱体积的Elution Buffer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。
1.4.3 中压层析柱法纯化
Co-NTA 亲和层析介质 6FF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Lysis Buffer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Wash Buffer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）。注：在样品和结合缓冲液中加入咪唑可以提高样品纯度。
⑥ 用Elution Buffer采用一步法或线性梯度洗脱。一步洗脱中，通常5倍柱体积洗脱液就足够了。梯度洗脱可以用一个小的梯度，例如20倍柱体积或更多，来分离不同结合强度的蛋白质。
上述步骤介质洗脱结束后，先用Lysis Buffer冲洗3倍柱体积，然后用纯水冲洗5倍柱体积，再用20%乙醇冲洗2个柱体积，然后将介质置于2–8℃保存。
1.5 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。
2. 在位清洗（CIP）
当填料使用过程中发现反压过高或者填料上面出现明显的污染时，需要进行在位清洗操作（Cleaning-in-Place，CIP）。建议按照下面操作去除填料上残留的污染物，如沉淀蛋白、疏水蛋白和脂蛋白等。
① 去除强疏水结合的蛋白、脂蛋白和脂类
通过使用30%异丙醇清洗5–10个柱体积，接触时间为15–20 min可以去除此类污染物。然后，再使用10倍柱体积的去离子水清洗。也可以选择使用含有去污剂的酸性或碱性溶液，清洗填料2倍柱体积。例如，含有0.1–0.5%非离子去污剂的0.1 M醋酸溶液，接触时间为1–2 h。去污剂处理后，需要使用70%的乙醇清洗5个柱体积，以彻底去除去污剂。最后使用10倍柱体积的去离子水清洗。
② 去除离子作用结合的蛋白
使用1.5 M NaCl溶液清洗10–15 min。然后，再使用去离子水清洗10个柱体积。清洗好的柱子可用20%乙醇冲洗2个柱体积，置于2–8℃保存。
3. 填料再生
组氨酸标签蛋白亲和纯化填料所带的钴离子不需要经常螯合去除和重新挂钴离子。当填料使用过程中发现颜色变浅，或者填料载量明显变低时，需要进行对填料进行钴离子剥离和重新挂钴离子，也就是填料再生。将填料装填在合适的层析柱内，按照下面操作流程进行钴离子剥离和重新挂钴离子。
① 使用5倍柱体积去离子水清洗填料；
② 使用5倍柱体积100 mM EDTA（pH 8.0）剥落钴离子；
③ 使用10倍柱体积去离子水清洗填料；
④ 使用0.5 M NaOH清洗5倍柱体积，停留10–15 min；
⑤ 使用去离子水清洗填料，直至pH中性；
⑥ 使用3–5倍柱体积100 mM CoCl₂再生挂钴；
⑦ 使用10倍柱体积去离子水清洗；
填料再生后，可以立即使用，如不立即使用，需要将填料悬浮于等体积的20%乙醇中，置于2–8℃保存。
Co-NTA Affinity Resin 6FF (His tag) enables one‑step purification of His‑tagged proteins from a variety of expression systems, such as E. coli, yeast, insect cells, and mammalian cells. The product is based on highly cross‑linked 6% agarose gel as the matrix, with cobalt ions chelated via tetra‑dentate coordination to the ligand. Owing to its pressure‑resistant matrix, it can withstand up to 0.3 MPa, making it particularly suitable for large‑scale industrial protein purification. Co-NTA Affinity Resin 6FF (His tag) also tolerates a range of denaturants and other additives (see Table 2). Another feature of Co-NTA Affinity Resin 6FF (His tag) is its higher selectivity for His‑tagged proteins compared to Ni-NTA Affinity Resin 6FF (His tag).
Aladdin’s Co-NTA Affinity Resin 6FF (His tag) is supplied as a 1:1 (v/v) slurry in 1× PBS containing 20% ethanol; the specified product volume refers to the actual settled gel volume.
Table 1. Performance properties of Co-NTA Affinity Resin 6FF (His tag)
| Parameter | Specification |
|---|
| Matrix | Highly cross‑linked 6% agarose | | Binding capacity | >20 mg 6×His‑tagged protein / ml medium | | Bead size | 45–165 μm | | Maximum pressure | 0.3 MPa, 3 bar | | Storage buffer | 1× PBS containing 20% ethanol | | Storage temperature | 2–8 °C |
|
Table 2. Reagent tolerance of Co-NTA Affinity Resin 6FF (His tag)
| Reagent type | Examples / concentrations |
|---|
| Reducing agents | 10 mM β‑mercaptoethanol① | | Denaturants | 8 M urea, 6 M Gua‑HCl | | Detergents | 1% Triton™ X‑100 (nonionic), 1% NP‑40 (nonionic), 1% CHAPS (zwitterionic), SDS, sarcosyl | | Other additives | 500 mM imidazole②, 30% ethanol③, 20% glycerol, 500 mM KCl, 1 M NaCl |
|
Notes:
① Do not store the medium in buffers containing β‑mercaptoethanol.
② Excessively high imidazole concentrations may reduce protein recovery.
③ Ethanol may cause protein precipitation, reduce recovery, and clog the column.
1. Purification Protocol
1.1 Buffer Preparation
The following recommended buffers can be used, or you may prepare different buffer systems according to your own preferences. The basic principle is loading at low imidazole concentration and eluting at high imidazole concentration. A neutral to slightly alkaline buffer (pH 7–8), such as phosphate buffer, is recommended. 0.3–0.5 M NaCl may be added to reduce non‑specific binding. Avoid using Tris‑HCl under conditions where protein binding is weak. Chelating agents such as EDTA and citrate buffers should be avoided. It is best to filter the buffers through a 0.22 μm or 0.45 μm membrane before use. Since Co-NTA Affinity Resin 6FF (His tag) can be used for both soluble protein and inclusion body protein purification, the buffers required differ between the two methods. Specific formulations are given in Table 3 and Table 4.
Table 3. Buffers and formulations for soluble His‑tagged protein purification
| Buffer Name | Volume | Formulation |
|---|
| Lysis Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl). Adjust pH to 8.0 with NaOH solution, filter through 0.22 or 0.45 μm membrane. | | Wash Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 5 mM imidazole (0.34 g imidazole). Adjust pH to 8.0 with NaOH solution, filter. | | Elution Buffer | 1 L | 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole). Adjust pH to 8.0 with NaOH solution, filter. |
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Table 4. Buffers and formulations for inclusion‑body His‑tagged protein purification (imidazole elution)
| Buffer Name | Volume | Formulation |
|---|
| Lysis Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl). Adjust pH to 8.0 with NaOH solution, filter. | | Wash Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole). Adjust pH to 8.0, filter. | | Elution Buffer | 1 L | 8 M Urea (480.50 g urea), 50 mM NaH₂PO₄ (7.80 g NaH₂PO₄·2H₂O), 300 mM NaCl (17.54 g NaCl), 250 mM imidazole (17.0 g imidazole). Adjust pH to 8.0, filter. |
|
1.2 Sample Preparation
1.2.1 Proteins expressed in bacteria or yeast
① Pick a single colony into culture medium. Add the appropriate concentration of inducer according to the vector instructions and induce for the appropriate time.
② After expression, transfer the culture to centrifuge bottles. Centrifuge at 7,000 rpm (7,500×g) for 15 min to collect the cells. Resuspend the cell pellet in Lysis Buffer at a ratio of 1:10 (W/V). Add PMSF to a final concentration of 1 mM. Add lysozyme (working concentration 0.2–0.4 mg/mL; if the host cells contain pLysS or pLysE, lysozyme may be omitted). Other protease inhibitors may also be added, provided they do not affect binding of the target protein to the medium.
③ Resuspend the cell pellet (if the cell density is high, consider adding 10 μg/mL RNase A and 5 μg/mL DNase I), mix well, and place on ice. Sonicate on ice until the cell suspension becomes essentially clear.
④ Transfer the clarified lysate to centrifuge tubes. Centrifuge at 10,000 rpm (15,000×g) for 20–30 min at 4 °C. Collect the supernatant and keep on ice for immediate use or store at –20 °C.
1.2.2 Soluble proteins secreted by yeast, insect, and mammalian cells
① Transfer the cell culture medium to centrifuge cups and centrifuge at 5,000 rpm (3,800×g) for 10 min to remove cells. If the supernatant does not contain EDTA, histidine, reducing agents, or similar substances, it can be applied directly to the column. If it contains such substances, dialyze against Lysis Buffer before loading.
② For large volumes of supernatant, concentrate by ammonium sulfate precipitation; the protein must then be dialyzed against Lysis Buffer before loading onto the column.
1.2.3 Inclusion body protein purification (denaturing conditions)
① Transfer the culture to centrifuge bottles and centrifuge at 7,000 rpm (7,500×g) for 15 min to collect the cells. Discard the supernatant.
② Resuspend the cells in Lysis Buffer (without 8 M urea) at a ratio of 1:10 (W/V), mix well, and sonicate on ice.
③ Transfer the lysate to centrifuge tubes and centrifuge at 10,000 rpm (15,000×g) for 20–30 min at 4 °C. Discard the supernatant. Steps 2 and 3 may be repeated once.
④ Resuspend the inclusion bodies in Lysis Buffer (containing 8 M urea) at a ratio of 1:10 (W/V).
⑤ Purify the His‑tagged protein under denaturing conditions using the buffer formulations given in Table 4.
1.3 Packing of Co-NTA Affinity Resin 6FF (His tag)
1.3.1 Packing a gravity column
① Take a gravity column of suitable size, insert the lower frit, rinse the column tube and frit with an appropriate amount of pure water, and close the bottom outlet.
② Mix the Co-NTA Affinity Resin 6FF (His tag) thoroughly. Using a pipette, transfer an appropriate volume of slurry into the gravity column (the actual settled medium volume is half of the slurry volume). Open the bottom outlet and allow the storage solution to drain.
③ Add an appropriate amount of pure water to wash the medium. After the liquid has drained by gravity, close the bottom outlet.
④ Insert the rinsed upper frit, ensuring that there is no gap between the frit and the resin bed and that the frit is level.
⑤ The packed gravity column can be equilibrated directly with equilibration buffer. If not used immediately, add storage solution and store at 2–8 °C.
1.3.2 Packing a medium‑pressure chromatography column
Co-NTA Affinity Resin 6FF (His tag) is widely used in industrial purification and is therefore packed into various medium‑pressure chromatography columns. The packing method is described below. Before packing, calculate the column base area from the column diameter, and calculate the required medium volume based on the desired packed bed height using the following formula:
V = 1.15 × π × r² × h
(V: required medium volume in mL; 1.15: compression factor; r: column radius in cm; h: packed bed height in cm)
Note: The volume of slurry taken should be twice the medium volume, because the settled medium accounts for only half of the total slurry volume; the other half is storage solution.
① Rinse the column bottom frit and adapter with deionized water, ensure no air bubbles remain on the bottom frit, close the bottom outlet, and leave 1–2 cm of deionized water in the column bottom.
② Resuspend the medium and carefully pour the slurry continuously into the column. Pouring the slurry along the column wall with a glass rod can help reduce bubble formation.
③ If a reservoir is used, immediately fill the column and the reservoir with water. Place the flow adapter on the slurry surface, connect to the pump, and avoid introducing air bubbles into the adapter or tubing.
④ Open the outlet at the bottom of the chromatography column and start the pump to run at the set flow rate. Initially, pass the buffer through the column at a low flow rate, then gradually ramp up to the final flow rate. This prevents hydraulic shock to the packed bed and avoids uneven bed formation.If the recommended pressure or flow rate cannot be reached, the maximum flow rate of your pump may be used, which can still give good packing results. (Note: In subsequent chromatography runs, do not exceed 75% of the maximum packing flow rate.) Once the bed height is stable, pass at least 3 column volumes of deionized water through the bed 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 adapter into the column.
⑦ Push the adapter down to the marked bed height. Allow packing liquid to enter the adapter, then lock the adapter fitting.
⑧ Connect the packed column to a pump or chromatography system and begin equilibration. The adapter can be readjusted if necessary.
1.4 Sample Purification Procedures
1.4.1 Batch incubation purification
① According to the sample volume, transfer an appropriate amount of Co-NTA Affinity Resin 6FF (His tag) to a centrifuge tube. Centrifuge at 1,000 rpm for 1 min and aspirate the supernatant. Alternatively, use a gravity column and allow the storage solution to drain.
② Add 5 medium volumes of Lysis Buffer to the centrifuge tube to wash the medium. Centrifuge at 1,000 rpm for 1 min and aspirate the supernatant. If using a gravity column, wash directly in the column by gravity flow. Repeat at least twice.
③ Add the sample, close the centrifuge tube or gravity column, and incubate with shaking at 4 °C for 2–4 h, or at 37 °C for 30 min–2 h.
④ After incubation, centrifuge at 1,000 rpm for 1 min and aspirate the supernatant, or filter to collect the medium. Retain the supernatant as the flow‑through fraction for electrophoretic analysis.
⑤ Wash the medium with 5 medium volumes of Wash Buffer. Centrifuge at 1,000 rpm for 1 min or filter through the gravity column, and remove the supernatant (be careful not to aspirate the medium). Repeat 3–5 times; it is recommended to transfer to a new centrifuge tube in between washes. The imidazole concentration in the wash buffer may be adjusted if necessary.
⑥ Add 3–5 column volumes of Elution Buffer and incubate at room temperature for 10–15 min. Centrifuge at 1,000 rpm for 1 min or collect the eluate from the gravity column. This elution step can be repeated 2–3 times.
1.4.2 Gravity column purification
① Equilibrate the packed Co-NTA Affinity Resin 6FF (His tag) gravity column with 5 column volumes of Lysis Buffer to bring the resin into the same buffer system as the target protein. Repeat 2–3 times.
② Apply the sample to the equilibrated gravity column. Allow a residence time of at least 2 min to ensure sufficient contact between the sample and the medium. Collect the flow‑through. The sample can be loaded repeatedly to increase binding efficiency.
③ Wash with 10–15 column volumes of Wash Buffer to remove non‑specifically bound contaminating proteins. Collect the wash fractions. The imidazole concentration in the wash buffer may be adjusted if necessary.
④ Elute with 5–10 column volumes of Elution Buffer. Collect fractions in separate tubes (one column volume per tube) and analyze each. This ensures that all bound target protein is eluted while also yielding protein of high purity and concentration.
1.4.3 Medium‑pressure column purification
After packing, Co-NTA Affinity Resin 6FF (His tag) can be used with various conventional medium‑ and low‑pressure chromatography systems.
① Fill the pump tubing with deionized water. Remove the top cap, connect the column to the chromatography system, open the bottom outlet, attach the prepacked column, and tighten the connections.
② Wash out the storage buffer with 3–5 column volumes of deionized water.
③ Equilibrate the column with at least 5 bed volumes of Lysis Buffer.
④ Load the sample using the pump or sample loop. Note: Increased sample viscosity can cause high backpressure even with small injection volumes. Do not exceed the binding capacity of the column. Large sample volumes may also cause high backpressure, making the injector more difficult to use.
⑤ Wash the column with Wash Buffer until the UV absorbance reaches a stable baseline (generally at least 10–15 column volumes). Note: Adding imidazole to the sample and binding buffer can improve sample purity.
⑥ Elute using Elution Buffer in either stepwise or linear gradient mode. For step elution, 5 column volumes of elution buffer are usually sufficient. For gradient elution, a shallow gradient (e.g., 20 column volumes or more) can be used to separate proteins with different binding strengths.
After elution in the above steps, wash the medium first with 3 column volumes of Lysis Buffer, then with 5 column volumes of pure water, and finally with 2 column volumes of 20% ethanol. Store the medium at 2–8 °C.
1.5 SDS‑PAGE Analysis
Analyze the purity of the purified product by SDS‑PAGE using the collected fractions (flow‑through, wash, and elution fractions) alongside the original sample.
2. Cleaning‑in‑Place (CIP)
When the resin shows increased backpressure or visible contamination during use, a Cleaning‑in‑Place (CIP) procedure should be performed. The following operations are recommended to remove residual contaminants such as precipitated proteins, hydrophobic proteins, and lipoproteins from the resin.
① Removal of strongly hydrophobically bound proteins, lipoproteins, and lipids
Wash with 30% isopropanol for 5–10 column volumes with a contact time of 15–20 min to remove such contaminants. Then wash with 10 column volumes of deionized water. Alternatively, use an acidic or alkaline solution containing detergent for 2 column volumes. For example, 0.1 M acetic acid containing 0.1–0.5% non‑ionic detergent, with a contact time of 1–2 h. After detergent treatment, wash with 5 column volumes of 70% ethanol to thoroughly remove the detergent. Finally, wash with 10 column volumes of deionized water.
② Removal of ionically bound proteins
Wash with 1.5 M NaCl solution for 10–15 min. Then wash with 10 column volumes of deionized water. The cleaned column can be rinsed with 2 column volumes of 20% ethanol and stored at 2–8 °C.
3. Resin Regeneration
The cobalt ions on the His‑tag affinity purification resin do not need to be stripped and recharged frequently. When the resin color becomes lighter or the binding capacity noticeably decreases during use, it is necessary to strip the cobalt ions and recharge them—this is resin regeneration. Pack the resin in a suitable chromatography column and follow the procedure below for cobalt stripping and recharging.
① Wash the resin with 5 column volumes of deionized water.
② Strip the cobalt ions with 5 column volumes of 100 mM EDTA (pH 8.0).
③ Wash the resin with 10 column volumes of deionized water.
④ Wash with 5 column volumes of 0.5 M NaOH and hold for 10–15 min.
⑤ Wash the resin with deionized water until the pH is neutral.
⑥ Recharge cobalt by washing with 3–5 column volumes of 100 mM CoCl₂.
⑦ Wash with 10 column volumes of deionized water.
After regeneration, the resin can be used immediately. If not used immediately, suspend the resin in an equal volume of 20% ethanol and store at 2–8 °C.
ZJ26F0737991
危险品化学品经营许可证（带存储）