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罗氏推出第一款蛋白亲和纯化预装柱:cOmplete His-Tag Purification Column
2013年08月08日 新产品 评论数 2 ⁄ 被围观 5,136+


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继罗氏推出第一款蛋白亲和纯化树脂:cOmplete His-Tag Purification Resin后,近日又推出了蛋白亲和纯化预装柱:cOmplete His-Tag Purification Column

灵活纯化蛋白、保证效能

选择靶蛋白适宜的缓冲条件

cOmplete His-Tag Purification Resin是一种新型的高效能IMAC基质,用于裂解液中His标签蛋白的一步法纯化,操作方便。采用罗氏特殊的镍螯合化学技术,该纯化树脂与常用的还原剂(如DTT )、金属蛋白酶螯合抑制剂(如E DTA)兼容,适用于广泛底物及盐浓度的缓冲条件。基于这种很强的组分兼容性,您可灵活地优化缓冲条件,以保持蛋白质的高度稳定性及溶解度,保护蛋白不被蛋白酶降解及避免被氧化。

使用DTT 与EDTA稳定您的蛋白。
灵活使用含E DTA或DTT 的缓冲液,在不降低纯化效能的情况下,保持蛋白的高度稳定性。

获得大量高纯度蛋白质。
树脂的高结合效能与结合特异性是一步法纯化蛋白的必备条件。

毒性废物排出显著降低。
镍脱落量极少,大大减少了毒性废物量,并避免了镍离子重新螯合填料的麻烦。

减少镍脱落

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 是通过一种特殊的化学技术,使用一种强效螯合剂将镍离子固定化获得的。

即使在E DTA与DTT 存在的条件下,镍离子的脱落量也极少,避免重新螯合填料的需要(图1)。

图1:严苛条件下,不同树脂的Ni 离子脱落情况。
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 ),对释放至缓冲液中的镍离子量进行测定。

结果:孵育期间,cOmplete His-Tag Purification Resin镍离子脱落率小于1%。而采用不同螯合剂的两种树脂,镍离子释放至缓冲液中的量分别高达76% 与59% 。

避免频繁的柱再生操作

随心所欲地进行靶蛋白合适的缓冲条件的选择,无需担心会降低树脂的稳定性。使用含还原剂(如DTT )与/ 或金属蛋白酶抑制剂(如E DTA)的缓冲液,不会改变cOmplete His-Tag Purification Resin的效能(图2 )。得益于新型螯合剂的作用,镍离子与树脂紧密结合(图2 )。

图2:无Ni 重新螯合的情况下,经多次使用后的树脂效能。在cOmplete His-Tag Purification Resin (0.5 ml)中装载含7ml His6-CFP (青色荧光蛋白)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。洗涤步骤后,使用1.5ml缓冲液B (缓冲液A+250 mM 咪唑)进行结合蛋白的洗脱,并对蛋白峰进行定量测定。在下一次纯化操作前,使用15ml缓冲液A进行树脂洗涤。对5次连续纯化操作后的 mAU 进行计算 (相当于柱纯化获得的总蛋白),绘制柱状图。

结果:即使在严苛的缓冲条件下,5 次蛋白纯化操作中,cOmplete His-Tag Purification Resin 依然可保持其稳定性,无需进行镍离子的更新螯合。

使用高效能树脂,获得高性价比

纯化柱对蛋白的结合效能主要取决于靶蛋白特性:如靶蛋白分子量、Stokes 半径、结合条件与添加组分。cOmplete His-Tag Purifcation Resin的设计是对经济性及高纯化性能的极佳平衡。在树脂中装载过量蛋白会导致蛋白质不稳定。cOmplete His-Tag Purification Resin对小分子蛋白的结合量最大。蛋白质分子越大,处理载量随之降低(表1)。

表1:树脂对各种蛋白的结合能力。

和其他任何树脂一样,cOmplete His-Tag Purification Resin 对蛋白的结合效能呈现时间依赖性。延长孵育时间可获得更高效能(图3)。

图3 :树脂结合蛋白的时间相关性。使用缓冲液A 进行250µl cOmplete His-Tag Purification Resin平衡处理,使用同等含量的His6-tagged T4 Gene 32蛋白溶液分别孵育10、20、30、45与60分钟。离心获得上清液,测定其中的蛋白质浓度。根据添加的蛋白量与上清中的蛋白量之间的差值进行已结合蛋白计算。

获得高度纯化蛋白

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标签蛋白的实验室分析及大规模纯化。通过粗裂解液的一步纯化处理,即可获得高纯度目标蛋白。仅单次纯化操作即可获得理想的蛋白纯度(图4)。

图4:His6-与His10-标签蛋白的有效纯化。2µl天然裂解液含有中等含量的His6-MBP(A)或His10-T4 DNA 连接酶(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成分为:150mM NaCl、50mM Tris-HCl pH 7.5 、2mM DTT 、5或10mM 咪唑。使用同样的缓冲液洗涤掉未结合组分,并使用附加400mM咪唑的缓冲液A进行蛋白洗脱。

结果:仅一次纯化操作,cOmplete His-Tag Purification Resin即可生成高度纯化的靶蛋白。

避免进行毒性镍溶液处理

Ni2+ 具有生物毒性,并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。镍的化学结构类似于锌,锌是多种酶的辅基,当镍占据锌结合位点时,酶功能将受到很高程度的影响。镍过量还可导致肺癌、鼻癌及骨肉瘤的发生。在cOmplete His-Tag Purification Resin 中,由于其使用的Ni 螯合剂的独特化学特性,树脂上结合的金属离子得到保护,防止还原剂的结合。因此,该纯化柱的镍脱落率降至极低,也不需要进行频繁的再生循环(包括使用镍溶液进行重新螯合),可显著降低毒性镍溶液处理的风险,降低有毒废液的处理成本与环境危害。

订购信息:
06781543001 5 columns (1 ml resin each)
06781535001 1 column (5 ml resin)

相关资料下载:

cOmplete His-Tag Purification Columns are available in 2 sizes and prepacked with 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.

cOmplete His-Tag Purification Resin

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.

 

cOmplete His-Tag Purification Resin, Protein-binding performance with His6 CFP cOmplete His-Tag Purification Resin, loss of resin Ni ions under stringent conditions
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.

 

 

 

 

cOmplete His-Tag Purification Resin, efficient purification of His6- and His10-tagged proteins Figure 4:
Efficient purification of His6- and His10-tagged proteins.

Two milliliters of native lysate containing moderate amounts of His6-MBP (A) or His10-T4 DNA Ligase (B), were incubated for 2 hours with 50 or 40 μl of cOmplete His-Tag Purification Resin in buffer A (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM DTT) containing 5 or 10 mM imidazole, respectively. Unbound material was washed off with the same buffers and eluted in buffer A supplemented with 400 mM imidazole.
Result: The cOmplete His-Tag Purification Resin produces highly pure target proteins after just one run.

MatrixSepharose-CL 6BBinding capacity> 40 mg protein per ml bed volume of resin
The binding capacity of the resin to various types of proteins may vary according to the protein characteristics such as the size of the protein.
cOmplete His-Tag Purification Columns bind with a high specificity to the polyhistidine-tagged protein. As a consequence, the binding kinetics may appear to be different when compared to conventional metal chelate matrices. Full capacity of cOmplete His-Tag Purification Columns can be achieved by allowing more time for the protein to bind to the resin by lowering the flow rate during the chromatography purification procedure.Maximal linear flow rate1,420 cm/hourRecommended volumetric flow rate5 ml column (06 781 535 001): 2.5 to 10 ml/minute
1 ml column (06 781 543 001): 0.5 to 2.0 ml/minute

The volumetric flow rate is a function of the column's cross section.Recommended imidazole concentration for load/washNonspecific binding of proteins without a His-tag is low.
Use up to 5 mM imidazole in load and/or wash buffers.

If establishing a new assay for purification of His-tag proteins with cOmplete His-Tag Purification Columns do not use imidazole. If, e.g., the purity of the His-tag protein needs to be improved following this first step, use imidazole in a final concentration of up to 5 mM in a second step.Recommended imidazole concentration for elutionUp to 500 mM

Please note:
In contrast to other available resins, bound His-tagged protein typically elutes from cOmplete His-Tag Purification Columns with a lower imidazole concentration, e.g., 25 to 45 mM.  Compatibility for long term storage20% ethanol, pH 4.0 to pH 9.0 Compatibility during chromatographyThe resin is compatible with 10 mM EDTA, 10 mM DTT during the purification (1 hour incubation), 6 M guanidinium-HCl, 8 M urea, pH 2.0 to pH 14.0. Compatibility during cleaning4% SDS Form cOmplete His-Tag Purification Resin filled in columns, pre-charged with Ni2+ stored in 20% ethanol.

Additional Information

Instructions for Use and Material Safety Data Sheets

Flyer
Reagents for Protein Expression and Analysis 
More Safety, Less Stress, Pure Protein

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Flyer (PDF, 1 MB)

Flyer
cOmplete His-Tag Purification Products 
Truly compatible with DTT and EDTA

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Flyer (PDF, 922 KB)

Protocol
cOmplete His-Tag Purification Products 
Quick Protocol

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Protocol (PDF, 500 KB)

Brochure
cOmplete His-Tag Purification Resin 
Truly compatible with DTT and EDTA

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Brochure (PDF, 2 MB)

 



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