S_0274_PROT_niNTA

✓ 연중무휴 하루 24시간 자동 온라인 주문 처리

✓ 풍부한 지식과 전문성을 갖춘 제품 및 기술 지원

✓ 신속하고 안정적인 (재)주문

Ni-NTA Superflow (25 ml)

Cat. No. / ID:  30410

25mL nickel-charged resin(최대 압력: 140psi)
₩681,000.00
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Volume
25 mL
100 mL
500 mL

✓ 연중무휴 하루 24시간 자동 온라인 주문 처리

✓ 풍부한 지식과 전문성을 갖춘 제품 및 기술 지원

✓ 신속하고 안정적인 (재)주문

특징

  • 우수한 기계적 안정성, 뛰어난 유동 특성, 높은 동적 결합력
  • 6x His-tagged 단백질을 하나의 단계에서 편리하게 정제
  • 효율적인 생산 규모 및 FPLC 응용 분야를 위한 높은 유속 및 압력 허용

제품 세부 정보

Ni-NTA Superflow는 His tag가 있는 재조합 단백질을 정제하는 데 사용하는 친화성 크로마토그래피 기질입니다. 

His tag의 히스티딘 잔류물은 고정화 니켈 이온의 배위 영역의 빈 위치에 높은 특이성과 친화성으로 결합됩니다. 투명 세포 용해물은 기질로 들어갑니다. His-tagged 단백질은 결합되고 다른 단백질은 기질을 통과합니다. His-tagged 단백질은 세척 후 기본 또는 변성 조건에서 완충액에 용출됩니다. 

성능

Ni-NTA Superflow는 Superflow 수지에 결합된 Ni-NTA로 구성됩니다. 이는 우수한 기계적 안정성, 뛰어난 유동 특성, 높은 동적 결합력을 겸비한 제품입니다. 6xHis-tagged 단백질 수용량은 5~10mg/mL입니다. 이 수지는 빠른 유속과 압력으로 6xHis-tagged 단백질을 한 단계로 정제할 수 있어 효율적인 생산 규모와 FPLC 응용 분야에 사용할 수 있습니다. 

 

 

원리

QIAexpress Ni-NTA Protein Purification System은 6개 이상의 히스티딘 잔류물의 친화성 태그(His tag)를 포함하는 단백질에 대한 특허받은 니켈-니트릴로트리아세트산(Nickel-Nitrilotriacetic Acid, Ni-NTA) 수지의 뛰어난 선택성을 기반으로 합니다. 이 기술을 사용하면 기본 또는 변성 조건의 모든 발현 시스템에서 거의 모든 His-tagged 단백질을 한 단계로 정제할 수 있습니다.  

니켈 이온에 대한 킬레이트화 부위가 네 개인 NTA는 금속 이온과 상호 작용할 수 있는 부위가 세 개뿐인 금속 킬레이트 정제 시스템보다 니켈과 더 단단히 결합합니다. 여분의 킬레이트화 부위는 니켈 이온 침출을 방지하여 다른 금속 킬레이트화 정제 시스템을 사용하여 얻은 것보다 결합력과 순도가 더 높은 단백질 제제를 생성합니다. Ni-NTA Superflow는 baculovirus, 포유류 세포, 효모, 박테리아를 포함한 모든 발현 시스템에서 His-tagged 단백질을 정제하는 데 사용할 수 있습니다.  

절차

His-tagged 단백질의 정제는 세포 용해, 결합, 세척, 용출의 4단계로 구성됩니다. Ni-NTA Superflow를 사용한 재조합 단백질의 정제는 단백질 또는 6xHis tag의 3차원 구조에 의존하지 않습니다. 따라서 기본 또는 변성 조건에서 희석액과 미가공 용해물에 있는 단백질을 한 단계로 정제할 수 있습니다.  

강력한 변성제와 세제는 수용체, 막 단백질과 봉입체를 형성하는 단백질을 효율적으로 용해하고 정제하는 데 사용할 수 있습니다. 비특이적으로 결합하는 오염 물질을 효율적으로 제거할 수 있는 시약을 세척 완충액에 포함할 수 있습니다(표 참조).  

정제된 단백질은 100~250mM 이미다졸을 경쟁 물질로 추가하거나 pH를 낮춰 온화한 조건에서 용출됩니다. 

응용 분야

Ni-NTA 기질은 His-tagged 단백질을 신뢰할 수 있는 한 단계로 정제하여 다음을 포함한 모든 응용 분야에 적합합니다. 

  • 구조 및 기능 조사 
  • 3차원 구조 결정을 위한 결정화 
  • 단백질-단백질 및 단백질-DNA 상호 작용 분석 
  • 항체 생성을 위한 면역화 
  • 정제를 생산 규모로 확장 

Ni-NTA Superflow의 경우, 규제 관련 문제를 지원하기 위해 제품 특성, 크로마토그래피 파라미터, 화학 및 물리적 정보, 품질 관리 사양, 안전성 및 독성 정보, 표준 정제 프로토콜이 포함된 원료의약품등록 파일(Drug Master File, DMF)을 제공합니다. 

 

지원되는 데이터 및 수치

Specifications

FeaturesSpecifications
Applications단백질체학
Scale대용량
Processing수동
Bead size60~160µm
FPLC
Special feature배치 및 컬럼 정제
Gravity flow or spin column중력류 또는 자동
Binding capacity최대 50mg/ml
Start material세포 용해물
Support/matrixSuperflow
Tag6xHis tag
Yield결합력에 따라 다름
Number of preps per run실험당 샘플 1~24개

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
Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.
Block H; Kubicek J; Labahn J; Roth U; Schäfer F;
Protein Expr Purif; 2007; 57 (2):244-54 2007 Oct 17 PMID:18053740
A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.
Chen CS; Korobkova E; Chen H; Zhu J; Jian X; Tao SC; He C; Zhu H;
Nat Methods; 2007; 5 (1):69-74 2007 Dec 16 PMID:18084297
Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor.
Yeliseev A; Zoubak L; Gawrisch K;
Protein Expr Purif; 2006; 53 (1):153-63 2006 Dec 12 PMID:17223358
Calbindin D(9k) knockout mice are indistinguishable from wild-type mice in phenotype and serum calcium level.
Kutuzova GD; Akhter S; Christakos S; Vanhooke J; Kimmel-Jehan C; Deluca HF;
Proc Natl Acad Sci U S A; 2006; 103 (33):12377-81 2006 Aug 8 PMID:16895982

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
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
Which resin is used in the QIAexpress Ni-NTA Fast Start Columns?
The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.
FAQ ID -836
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
Should I use Ni-NTA Agarose in column or batch format for purification of 6xHis-tagged proteins?
The binding capacity of Ni-NTA Agarose is the same regardless of the format used. However, the batch procedure (mixing the Ni-NTA resin with lysate or protein sample prior to loading it onto a column, as opposed to loading the sample onto a column pre-packed with Ni-NTA resin) can provide more efficient binding for dilute proteins, since binding can be carried out for an extended period (approximately 1 hour), and resin amounts can be scaled for variable amounts of lysate/protein sample.
FAQ ID -147
What is the difference between Ni-NTA Agarose and Ni-NTA Superflow?

The binding capacity of both resins is the same: up to 50mg/ ml mg 6xHis-tagged protein per ml of resin (2500 nmol @ ~20 kDa). The difference between them is the bead support, which determines pressure resistance and flow rate:

Ni-NTA Agarose:

  • Sepharose CL-6B (bead size 45–165 µm)
  • max. volumetric: 0.5–1.0 ml/min
  • max. pressure: 2.8 psi/(0.2bar)
  • for use with gravity flow only

Ni-NTA Superflow:

  • Superflow (bead size 60–160 µm)
  • max. volumetric: 20 ml/min
  • max. pressure: 140 psi/(10bar)
  • for use with gravity flow or FPLC

You can find a detailed comparison table in the Appendix at the back of the QIAexpressionist Handbook under the title 'Ni-NTA Matrices'.

FAQ ID -764
Can I reuse the Ni-NTA Agarose and Ni-NTA Superflow resins?

The reuse of Ni-NTA Agarose and Ni-NTA Superflow resins depends on the nature of the sample and should only be performed with identical recombinant proteins. We recommend a maximum of 5 runs per column. After use the resin should be washed for 30 minutes with 0.5 M NaOH. Ni-NTA matrices should be stored in 30% ethanol to inhibit microbial growth.

If the Ni-NTA matrix changes from light blue to brownish-gray, the regeneration procedure described in the Appendix of the QIAexpressionist Handbook in section 'Reuse of Ni-NTA Resin' is recommended.

FAQ ID -802
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
Do you have a protocol for manual purification of 6xHis-tagged proteins expressed in E. coli using Ni-NTA Superflow?
How can I check if any residual proteins remain on the Ni-NTA Agarose matrix after elution?
Ni-NTA Agarose may be boiled in SDS-PAGE sample buffer to release any protein that remains on the matrix following elution. All proteins, regardless of whether they bind to Ni-NTA or to the agarose-moiety, will be recovered by this procedure.
FAQ ID -324
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