继罗氏推出第一款卵白亲和纯化树脂：cOmplete His-Tag Purification Resin后沈先生 探花，近日又推出了卵白亲和纯化预装柱：cOmplete His-Tag Purification Column
天真纯化卵白、保证效率
遴选靶卵白合适的缓冲要求
cOmplete His-Tag Purification Resin是一种新式的高效率IMAC基质，用于裂解液中His标签卵白的一步法纯化，操作浮浅。遴选罗氏罕见的镍螯合化学时期，该纯化树脂与常用的规复剂（如DTT ）、金属卵白酶螯合扼制剂（如E DTA）兼容，适用于粗犷底物及盐浓度的缓冲要求。基于这种很强的组分兼容性，您可天真地优化缓冲要求，以保执卵白质的高度表示性及融解度，保护卵白不被卵白酶降解及幸免被氧化。
使用DTT 与EDTA表示您的卵白。 天真使用含E DTA或DTT 的缓冲液，在不裁减纯化效率的情况下，保执卵白的高度表示性。
获取无数高纯度卵白质。 树脂的高统一效率与统一特异性是一步法纯化卵白的必备要求。
毒性废料排出显赫裁减。 镍零碎量一丝，大大减少了毒性废料量，并幸免了镍离子再行螯合填料的艰辛。
减少镍零碎
与氨基三乙酸（NTA ）树脂或亚氨基二乙酸（IDA ）螯合树脂不同，cOmplete His-Tag Purification Resin 是通过一种罕见的化学时期，使用一种强效螯合剂将镍离子固定化获取的。
即使在E DTA与DTT 存在的要求下，镍离子的零碎量也一丝，幸免再行螯合填料的需要（图1）。
图1：严苛要求下，不同树脂的Ni 离子零碎情况。 cOmplete His-Tag Purification Resin 与另两种不同螯合剂树脂在9倍体积的含10 mM EDTA 、10 mM DTT、500 mM 咪唑、300 mM NaCl与50 mM NaH2PO4的缓冲液中（pH8.0），室温孵育1 小时。通过电感耦合等离子体质谱法（ICP-MS ），对开释至缓冲液中的镍离子量进行测定。
恶果：孵育时间，cOmplete His-Tag Purification Resin镍离子零碎率小于1%。而遴选不同螯合剂的两种树脂，镍离子开释至缓冲液中的量辞别高达76% 与59% 。
好利来 丝袜幸免正常的柱再生操作
运用自若地进行靶卵白合适的缓冲要求的遴选，无需系念会裁减树脂的表示性。使用含规复剂（如DTT ）与/ 或金属卵白酶扼制剂（如E DTA）的缓冲液，不会改造cOmplete His-Tag Purification Resin的效率（图2 ）。收获于新式螯合剂的作用，镍离子与树脂雅致统一（图2 ）。
图2：无Ni 再行螯合的情况下，经屡次使用后的树脂效率。在cOmplete His-Tag Purification Resin (0.5 ml)中装载含7ml His6-CFP （青色荧光卵白）的裂解液。裂解缓冲液含有 10mM EDTA 与10mM DTT 。使用5ml 缓冲液A （50mM NaH2PO4 pH 8.0/RT 、300mM NaCl 、10mM EDTA、10mM DTT ）洗涤树脂。洗涤尺度后，使用1.5ml缓冲液B （缓冲液A+250 mM 咪唑）进行统一卵白的洗脱，并对卵白峰进行定量测定。不才一次纯化操作前，使用15ml缓冲液A进行树脂洗涤。对5次连气儿纯化操作后的 mAU 进行计较 （十分于柱纯化获取的总卵白），欧美性爱图片绘画柱状图。
恶果：即使在严苛的缓冲要求下，5 次卵白纯化操作中，cOmplete His-Tag Purification Resin 已经可保执其表示性，无需进行镍离子的更新螯合。
使用高效率树脂，获取高性价比
纯化柱对卵白的统一效率主要取决于靶卵白特质：如靶卵白分子量、Stokes 半径、统一要求与添加组分。cOmplete His-Tag Purifcation Resin的研讨是对经济性及高纯化性能的极佳均衡。在树脂中装载过量卵白会导致卵白质不表示。cOmplete His-Tag Purification Resin对小分子卵白的统一量最大。卵白质分子越大，贬责载量随之裁减（表1）。
表1：树脂对多样卵白的统一智商。
和其他任何树脂一样，cOmplete His-Tag Purification Resin 对卵白的统一效率呈刻下辰依赖性。延伸孵育时辰可获取更高效率（图3）。
图3 ：树脂统一卵白的时辰相干性。使用缓冲液A 进行250µl cOmplete His-Tag Purification Resin均衡贬责，使用同等含量的His6-tagged T4 Gene 32卵白溶液辞别孵育10、20、30、45与60分钟。离心获取上清液，测定其中的卵白质浓度。凭证添加的卵白量与上清中的卵白量之间的差值进行已统一卵白计较。
获取高度纯化卵白
cOmplete His-Tag Purification Resin是一种基于Sepharose琼脂糖、预带电荷的、即用型N i2+- 螯合基质，用于His标签卵白的本质室分析及大范畴纯化。通过粗裂解液的一步纯化贬责，即可获取高纯度主义卵白。仅单次纯化操作即可获取理念念的卵白纯度（图4）。
图4：His6-与His10-标签卵白的有用纯化。2µl自然裂解液含有中等含量的His6-MBP（A）或His10-T4 DNA 纠合酶（B），将裂解液与缓冲液A，及50或40µl cOmplete His-Tag Purification Resin共孵育2小时，缓冲液A要素为：150mM NaCl、50mM Tris-HCl pH 7.5 、2mM DTT 、5或10mM 咪唑。使用雷同的缓冲液洗涤掉未统一组分，并使用附加400mM咪唑的缓冲液A进行卵白洗脱。
恶果：仅一次纯化操作，cOmplete His-Tag Purification Resin即可生成高度纯化的靶卵白。
幸免进行毒性镍溶液贬责
Ni2+ 具有生物毒性，而且是一种已知致癌剂。镍对带有氧、氮与硫供体的配基具有高度亲和性，因此会影响酶及DNA的功能。镍的化学结构访佛于锌，锌是多种酶的辅基，当镍占据锌统一位点时，酶功能将受到很高经过的影响。镍过量还可导致肺癌、鼻癌及骨赘瘤的发生。在cOmplete His-Tag Purification Resin 中，由于其使用的Ni 螯合剂的特有化学特质，树脂上统一的金属离子得到保护，详确规复剂的统一。因此，该纯化柱的镍零碎率降终点低，也不需要进行正常的再生轮回（包括使用镍溶液进行再行螯合），可显赫裁减毒性镍溶液贬责的风险，裁减有毒废液的贬责资本与环境危害。
订购信息： 06781543001 5 columns (1 ml resin each) 06781535001 1 column (5 ml resin)
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cOmplete His-Tag Purification Columns are available in 2 sizes and prepacked with 沈先生 探花cOmplete His-Tag Purification Resin. The cOmplete His-Tag Purification Resin is an innovative high-capacity IMAC matrix (Immobilized Metal Affinity Chromatography) for convenient single-step purifications of His-tagged proteins from total lysates. Roche's propriety nickel-chelate chemistry ensures extraordinary compatibility with commonly used reducing agents such as DTT， chelating metalloprotease inhibitors such as EDTA， and a wide range of buffer substances and salt conditions. The wide choice of compatible ingredients allows optimization of buffers for maximum protein stability and solubility. cOmplete His-Tag Purification Columns are fully compatible with standard purification systems such as ÄKTA Systems (GE Healthcare).
Use the buffer conditions best suited to your protein Keep your protein comfortable and let it， not your purification resin， determine whether you use DTT， EDTA， or other buffer substances. Repeatedly obtain highly pure protein Single step purification without resin recharging. Protect your protein from toxic nickel Reduce protein oxidation and aggregation caused by resins that leach nickel. Work in a safe and eco-friendly environment Avoid handling of toxic nickel and completely eliminate disposal costs.
Request your sample here and discover how efficiently this new resin purifies your His-tagged proteins – both as loose resin and as pre-packed column. Combine cOmplete His-Tag Purification Resin and Columns with cOmplete ULTRA Tablets or cOmplete Tablets containing EDTA without loss of capacity or purity.
Get to know the people behind our products in the Roche Tips from the developer series.
See the Quick Protocol for optimization tips with cOmplete His-tag Purification Products.
Recombinant Protein Expression
Purifying a protein of interest is often essential for determining its function， structure， or interactions， for raising specific antibodies， or preparing enzymes for practical applications. Isolation of naturally expressed proteins from their original source can be a complex process involving numerous chromatographic steps. Recombinant protein expression in dedicated host organisms can greatly simplify this task. Such expression systems generally ensure higher expression levels. Fusing the target protein to a tag also confers advantageous binding ability to an affinity matrix.
Protein Purification using Immobilized Ni2+
The most common technique for efficiently obtaining large yields of highly purified protein in a short timeframe involves engineering a polyhistidine tag into the protein of interest， followed by purification using Immobilized Metal Ion Affinity Chromatography (IMAC). The most commonly used tag for large amounts of highly purified protein is a polyhistidine tag (His-tag). This tag has 6 to 14 histidines， typically fused to the N- or C-terminal end of a target protein. In some cases， the tag is also inserted into an exposed loop of the target protein. The imidazole side chains of a His-tag can form reversible coordinative bonds to divalent metal ions， such as Ni2+， Co2+， or Zn2+. This property can be used to separate polyhistidine-tagged target proteins from other proteins. Ni2+ show highest affinity and selectivity for His-tags， and are therefore the preferred ions. Using a specific chelator covalently linked to a matrix， Ni2+ are immobilized to still permit interactions with histidine side chains. When His-tagged proteins are applied to such a Ni2+ resin， they specifically bind to the resin via Ni2+， while most untagged proteins do not. Bound proteins are released from the resin using mild conditions. Imidazole competes for coordination sites on Ni2+ and therefore displaces His-tagged proteins from the resin. Alternatively， lowering the pH will protonate His-tags， decreasing their affinity for the resin and hence elute the His-tagged proteins.
His-Tags
Ideally， the His-tagged target protein binds much stronger to the Ni2+ chelate matrix than endogenous histidine-containing protein of the expression host. Relative binding strength depends on how many histidines can bind simultaneously to the matrix (avidity effect). Longer His-tags confer stronger binding and better separation of the target from potentially contaminating host proteins. The classic His-tag has six consecutive histidines. Tags with 10 to 14 histidines may produce a better purification. Most importantly， His-tagged proteins can be purified using Ni2+ chelate matrices under both native and denaturing conditions. Due to their hydrophilic and flexible nature， these matrices increase the solubility of the target proteins and only rarely interfere with protein function. This unique combination of features enables the His-tag to be a versatile tool for a wide range of protein purification applications.
Figure 1: Tight binding of nickel visualized. Before (left) and after (right) photos of cOmplete His-Tag Purification Resin and Columns after 5 times reuse without recharging. The resin remains blue both before and after reusing， indicating that there is little to no nickel leakage.
Figure 2: Protein-binding performance with His6 CFP. cOmplete His-Tag Purification Resin (blue columns) with 10 mM DTT and EDTA is reused without nickel recharging alongside Resin G (grey columns) with 1 mM EDTA and 5 mM DTT ( as specified in manufactor's package insert). Another competing product， Resin Q (not shown)， did not bind any protein at all. Figure 3: Loss of resin Ni ions under stringent conditions. One milliliter each of cOmplete His-Tag Purification Resin and 2 commercially available resins were incubated in 9 ml of a buffer containing 50 mM NaH2PO4， 300 mM NaCl， pH 8.0， 10 mM EDTA， 10 mM DTT， and 500 mM imidazole. The cOmplete Resin lost less than 1 percent of nickel ions.