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Ni-NTA Spin Kit (50)

Cat. No. / ID:  31314

Ni-NTA Spin Columns 50개, 시약, 완충액, 콜렉션 튜브, 대조군 발현 플라스미드 1μg
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KitColumn
Ni-NTA Spin Kit
Ni-NTA Spin Column
Ni-NTA Spin System은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품들은 질병의 진단, 예방, 또는 치료 목적으로 사용할 수 없습니다.

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✓ Knowledgeable and professional Product & Technical Support

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Features

  • 단 15분 내 컬럼당 최대 300μg의 His-tagged 단백질 정제
  • 네이티브 및 변성 조건에서의 정제
  • 한 단계로 최대 95%의 균질성 달성
  • 신속한 자동 또는 수동 처리를 위한 즉시 사용 가능한 스핀 컬럼

Product Details

Ni-NTA 실리카는 비특이적 소수성 상호작용을 억제하도록 최적화된 거대 다공성 실리카 지지 물질과 Ni-NTA를 결합한 것입니다. Ni-NTA Spin Columns(His-단백질 정제 스핀 컬럼)는 Ni-NTA Spin Kit에 포함되어 있고, 별도로 구입 가능하며, 편리한 마이크로 스핀 형식의 Ni-NTA 실리카를 제공하여 여러 샘플을 병렬적으로 쉽게 준비할 수 있도록 합니다. 엔지니어링 단백질의 기능 스크리닝, 전장 번역 산물을 발현하는 클론 선택, 발현 수준 비교를 위한 간단한 방법을 제공합니다. 각 스핀 컬럼은 최대 300µg의 His-tagged 단백질을 정제할 수 있습니다. 모든 Ni-NTA 매트릭스와 마찬가지로 Ni-NTA 스핀 컬럼은 네이티브 또는 변성 조건에서 원스텝 단백질 정제에 사용할 수 있습니다. Ni-NTA Spin Kit는 His-tagged 단백질의 스핀 정제를 위한 완벽한 키트입니다. It can be automated on the QIAcube Connect (see image " QIAcube Connect").

See figures

Performance

Ni-NTA Spin Columns(His-단백질 정제 스핀 컬럼)도 Ni-NTA Spin Kit에 포함되어 있어 다양한 발현 수준에서(그림  '다양한 발현 수준에서 정제' 참조) 재현 가능한 빠른 자동 정제가 가능합니다(그림  '재현 가능한 자동 정제' 참조).

See figures

Principle

Ni-NTA Spin Columns 및 Ni-NTA Spin Kit를 포함한 QIAexpress Ni-NTA Protein Purification System은 6개 이상의 히스티딘 잔기인 친화성 태그인 His tag를 포함하는 단백질에 대한 특허받은 Ni-NTA(니켈-니트로트리아세트산) 수지의 뛰어난 선택성을 기반으로 합니다. 이 기술을 사용하면 네이티브 또는 변성 조건하의 모든 발현 시스템에서 거의 모든 His-tagged 단백질의 원스텝 정제가 가능합니다. 니켈 이온의 킬레이트화 부위가 4개인 NTA는 금속 이온과 상호작용할 수 있는 부위가 3개밖에 없는 금속 킬레이트 정제 시스템보다 니켈을 더 단단히 결합합니다. 추가 킬레이트화 부위는 니켈 이온 침출을 방지하고 다른 금속 킬레이트화 정제 시스템을 사용하여 얻은 것보다 더 큰 결합 용량과 더 높은 순도의 단백질 제제를 생성합니다. QIAexpress 시스템은 배큘로바이러스, 포유류 세포, 효모 및 박테리아를 포함한 모든 발현 시스템에서 His-tagged 단백질을 정제하는 데 사용할 수 있습니다.

Procedure

His-tagged 단백질의 정제는 세포 용해, 결합, 세척 및 용출의 4단계로 구성되어 있습니다(그림 'Ni-NTA 단백질 정제 시스템을 사용한 Ni-NTA Spin Column 정제' 참조). QIAexpress 시스템을 사용한 재조합 단백질의 정제는 단백질의 3차원 구조 또는 His tag에 의존하지 않습니다. 이를 통해 희석 용액과 조잡한 용해물로부터 원시 또는 변성 조건에서 원스텝 단백질 정제가 가능합니다. 최대 600μL의 세포 용해물을 Ni-NTA 스핀 컬럼에 로드합니다. 2분간 빠르게 회전하면 tagged 단백질은 Ni-NTA 실리카에 결합하고 태그되지 않은 단백질은 대부분 통과합니다. 세척 단계 후 정제된 단백질은 100~300μL 부피의 온화한 조건(pH를 5.9로 낮추거나 100~500mM 이미다졸을 첨가하는 등)에서 용출됩니다. His tag는 크기가 작고 면역 원성이 거의 없기 때문에 일반적으로는 제거할 필요가 없습니다. 정제된 단백질은 즉시 사용할 수 있습니다. 여러 소규모 발현 배양에서 단백질을 약 30분(수동 절차) 또는 약 60분(자동화된 QIAcube Connect 절차) 내에 정제할 수 있습니다. 수용체, 막 단백질, 봉입체를 형성하는 단백질의 효율적인 용해 및 정제를 위해 강력한 변성제 및 세제를 사용할 수 있습니다. 비특이적으로 결합하는 오염 물질을 효율적으로 제거할 수 있는 시약을 세척 완충액에 첨가할 수 있습니다(표 참조). 정제된 단백질은 경쟁 물질로 100~250mM 이미다졸을 첨가하거나 pH를 낮추어 온화한 조건에서 용출됩니다.

Ni-NTA–His 상호 작용과 호환되는 시약:

  • 6M 구아니딘 HCl
  • 8M 요소
  • 2% Triton X-100
  • 2% Tween 20
  • 1% CHAPS
  • 20mM β-ME
  • 10mM DTT
  • 50% 글리세롤
  • 20% 에탄올
  • 2M NaCl
  • 4M MgCl2
  • 5mM CaCl2
  • ≤20mM 이미다졸
  • 20mM TCEP
See figures

Applications

Ni-NTA Spin Columns 및 Ni-NTA Spin Kit를 포함한 QIAexpress Ni-NTA Protein Purification System은 다음을 비롯한 모든 애플리케이션에 적합한 신뢰할 수 있는 원스텝 단백질 정제를 제공합니다.

  • 구조적 및 기능적 조사
  • 3차원 구조 결정을 위한 결정화
  • 단백질–단백질 및 단백질–DNA 상호작용 관련 분석
  • 항체 생성을 위한 예방접종

 

Ni-NTA Spin Columns와 Ni-NTA Spin Kit 비교
특성 Ni-NTA Spin Columns Ni-NTA Spin Kit
애플리케이션 단백질체학 단백질체학
비드 크기 16–24 µm 16–24 µm
결합 용량 스핀 컬럼당 최대 300µg 스핀 컬럼당 최대 300µg
중력류 또는 스핀 컬럼 스핀 컬럼 스핀 컬럼
처리 자동/수동 자동
규모 소규모 소규모
특수 기능 처리량이 적은 스크리닝 한 단계로 최대 95%의 균질성 달성
시작 물질 세포 용해물 세포 용해물
지원/매트릭스 거대 다공성 실리카 거대 다공성 실리카
태그 6xHis tag 6xHis tag

Supporting data and figures

Publications

A highly specific system for efficient enzymatic removal of tags from recombinant proteins.
Schäfer F; Schäfer A; Steinert K;
J Biomol Tech; 2002; 13 (3):158-71 2002 Sep PMID:19498979

FAQ

What are your recommendations for PCR template preparation for use with the EasyXpress Insect Kit II?

We recommend to use the EasyXpress Linear Template Kit Plus to generate PCR products optimized for use in protein expression with the EasyXpress Insect Kit II.

This kit uses specially designed primers to amplify coding DNA sequence and supplement it with regulatory elements required for optimal transcription and translation in cell-free expression systems. In addition, specially designed 5' untranslated regions (UTRs) on the sense adapter primer sequences reduce the formation of secondary structure in the translation initiation region, one of the commonest causes of low expression rates. A His-or Strep-tag II can be added to either terminus, greatly simplifying protein purification and detection after expression.

FAQ ID -1221
Are the buffers in the Ni-NTA Fast Start Kit the same as the ones for use with Ni-NTA purchased separately?

The buffers of the Ni-NTA Fast Start Kit are based on recipes for the respective buffers for purification of 6xHis-tagged proteins under native or denaturing conditions listed in the QIAexpressionist handbook. Specific components have been added for optimized performance. The exact composition of the buffers in the Ni-NTA Fast Start Kit is confidential. However, the buffers listed in the Appendix Section of the QIAexpressionist are compatible with the Ni-NTA Fast Start Kit, and can also be used.

FAQ ID -791
How do I prevent bubbles from forming in my Ni-NTA agarose column?
Gas bubbles may form when the resin undergoes a temperature change. To alleviate this problem, degas the Ni-NTA agarose in equilibration buffer or simply keep the agarose at a constant temperature to keep all the gases in solution.
FAQ ID -285
Is it possible to isolate both RNA and recombinant 6xHis-tagged protein from the same sample?
We have no experimental data for this application. However, buffer RLT of the RNeasy Kits for RNA isolation does not contain substances incompatible with Ni-NTA purification of His-tagged proteins. It should be possible to first extract RNA from a sample by following the RNeasy procedure, save the flow-through from the binding step as well as from the RW1 wash, and apply the combined fractions onto a Ni-NTA column for binding of His-tagged proteins. Follow our recommendations for purification of 6xHis-tagged proteins using Ni-NTA resins outlined in the QIAexpressionist handbook.
FAQ ID -532
How can I remove imidazole from a protein sample?
Imidazole does not interfere with most downstream applications and therefore does not need to be removed. If it is necessary to remove the imidazole (e.g., for some sensitive enzyme assays), it can be easily achieved by dialysis, precipitation (e.g., ammonium sulfate), or ultrafiltration.
FAQ ID -91
What are the features and benefits of the QIAexpress 6xHis Tag System?

FEATURES BENEFITS
The interaction of the 6xHis tag with Ni-NTA matrices is conformation independent One-step purification can be carried out under native or denaturing conditions
Mild elution conditions can be used Binding, washing, and elution are highly reproducible, and have no effect on protein structure. Pure protein products are ready for direct use in downstream applications
The 6xHis tag is much smaller than other commonly used tags 6xHis tags can be used in any expression system. The Tag does not interfere with the structure and function of the recombinant protein
The 6xHis tag is uncharged at physiological pH The 6xHis tag does not interfere with secretion
The 6xHis tag is poorly immunogenic The recombinant protein can be used without prior removal of the tag as an antigen to generate antibodies against the protein of interest
Using Factor Xa Protease, 6xHis tag can be easily and efficiently removed The detagged protein can be used for crystallographical or NMR studies where removal of the 6xHis tag may be preferred
Some QIAexpress vectors feature a 6xHis-dihydrofolate reductase tag (6xHis-DHFR tag) Small peptides fused to the 6xHis DHFR tag are stabilized while being expressed. The 6xHis-DHFR tag is not highly immunogenic in mouse and rat, so that peptides fused to the tag can be used directly for immunizations or epitope mapping

 

FAQ ID -193
Can I use HEPES buffer instead of phosphate in my Ni-NTA column?
Although we do not strongly recommend it, up to 100 mM has been successfully used in some cases. Buffers that contain secondary or tertiary amines will reduce nickel ions and affect binding of 6xHis tagged proteins to the Ni-NTA resin.
FAQ ID -291
3354 - What type and amount of resin is packed into the Ni-NTA Spin columns from the Ni-NTA Spin Kit?

The Ni-NTA Spin columns used in the Ni-NTA Spin Kit contain a slightly different resin type than our other Ni-NTA columns. The columns in the Spin Kit contain approximately 25 mg of Ni-NTA Silica.

FAQ ID - 3354
How does imidazole affect my quantitation of protein?

Since imidazole absorbs UV radiation at 280 nm, an elution profile measured at 280 nm while purifying a 6xHis tagged protein by FPLC will show an increase in absorbance above the background signal allowing quantitation of your protein. The absorbance of imidazole can vary depending on its source and purity, but elution buffer containing 250 mM imidazole usually has an A280 of 0.2–0.4.

To quantitate proteins in eluates containing imidazole, we recommend the Bradford protein assay, which is based on the ability of Coomassie Brilliant Blue R250 to form strong complexes with proteins. This assay is more tolerant of higher imidazole concentrations than the Lowry and the biuret assay, which are more sensitive to imidazole because they involve the reduction of copper. For accurate measurements, always use elution buffer without protein as the reagent blank.

FAQ ID -132
Can Ni-NTA resins be used to purify protein with an internal His-tag?
Yes, Ni-NTA Agarose and Superflow will bind a 6xHis-tag whether it is located internally or at the C- or N-teminal end of the protein. Note that the His-tag must be exposed for binding at the surface of the protein to allow for efficient purification under native conditions.
FAQ ID -496
What are the compatibilities of different reagents with Ni-NTA matrices?

Compatibility of reagents with Ni-NTA matrices

Reagent Effect Comments
Buffer reagents    
Tris, HEPES, MOPS Buffers with secondary or tertiary amines will reduce nickel ions

Up to 100 mM has been used successfully in some cases

Sodium phosphate or phosphate-citrate buffer is recommended

Chelating reagents    
EDTA, EGTA Strip nickel ions from resin Up to 1 mM has been used successfully in some cases, but care must be taken
Sulfhydril reagents    
beta-mercaptoethanol Prevents disulfide cross-linkages Up to 20 mM
DTT, DTE Low concentrations will reduce nickel ions A maximum of 1 mM may be reduce nickel ions used, but beta-mercaptoethanol is recommended
Detergents    
Nonionic detergents (Triton, Tween, NP-40, etc.) Removes background proteins and nucleic acids Up to 2% can be used
Cationic detergents   Up to 1% can be used
CHAPS   Up to 1% can be used
Anionic detergents (SDS, sarkosyl)   Not recommended, but up to 0.3% has been used success-fully in some cases
Denaturants Solubilize proteins  
GuHCl   Up to 6 M
Urea   Up to 8 M
Amino acids    
Glycine   Not recommended
Glutamine   Not recommended
Arginine   Not recommended
Histidine Binds to Ni-NTA and competes with histidine residues in the 6xHis tag Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged protein from the Ni-NTA matrix
Other additives    
NaCl Prevents ionic interactions Up to 2 M can be used, at least 300 mM should be used
MgCl2   Up to 4 M
CaCl2   Up to 5 mM
Glycerol Prevents hydrophobic interaction between proteins Up to 50%
Ethanol Prevents hydrophobic interactions between proteins Up to 20%
Imidazole Binds to Ni-NTA and competes with histidine residues in the 6xHis tag Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged
Sodium bicarbonate   Not recommended

Hemoglobin

 

Ammonium

 

Citrate

 

Not recommended

 

Not recommended

 

Up to 60mM has been used successfully

 

 

FAQ ID -49
How can I avoid poor immunolocalization morphology with Anti-His Antibodies?
Insufficient fixation of samples leads to poor morphological preservation. If using methanol or acetone try fixation with paraformaldehyde. If 2% paraformaldehyde is insufficient, increase fixative concentration to 4% and increase incubation time. Keep in mind that too harsh fixation can lead to functional alteration of the 6xHis epitope and loss of recognition by the Anti·His Antibodies.immunofluorescence
FAQ ID -200
How can I improve the expression of proteins containing hydrophobic regions?
The addition of either 1% Octylglycoside or 1% Dodecylmaltoside to the lysate might help to solubilize proteins containing large hydrophobic regions. The detergents should be added directly to the reaction mix.
FAQ ID -339
How can I eliminate contaminating protein in my Ni-NTA 6xHis-tag protein purification?
  • Use 10-20 mM imidazole in the lysis and wash buffers (both for native and denaturing conditions). Optimal imidazole concentrations have to be determined empirically.
  • Increase the NaCl concentration (up to 2 M) in the purification buffers to reduce the binding of contaminants as a result of nonspecific ionic interactions.
  • Add ß-mercaptoethanol (up to 20 mM) to the lysis buffer to prevent copurification of host proteins that may have formed disulfide bonds with the protein of interest during cell lysis.
  • Add detergents such as Triton X-100 and Tween 20 (up to 2%), or additives such as glycerol (up to 50%) or ethanol (up to 20%) to reduce nonspecific binding to the matrix due to nonspecific hydrophobic interactions.
  • Reduce the amount of Ni-NTA matrix. Low-affinity binding of background proteins will be reduced by matching the total binding capacity of Ni-NTA matrix with the expected amount of 6xHis-tagged protein.
FAQ ID -102
Why do you recommend using Triton X for the purification of 6xHis-tagged protein?

Nonionic detergents such as Triton X-100 (0.1 - 1%) and Tween 20 (up to 2%) can be used to reduce non-specific binding of contaminating proteins due to non-specific hydrophobic or ionic interactions. They will have no effect on the binding of 6xHis-tagged protein to the Ni-NTA resin when used within the recommended concentration range.

Optimal concentrations for these additives to binding and wash buffers should be determined empirically for each purification protocol and protein.

-100
How can I be sure that I am harvesting my induced bacterial culture at the best time point for protein expression?

To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.

Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."

 

 

 

Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.

 

 

FAQ ID -788