Ni-NTA Fast Start Kit

E. coli 용해물에 있는 재조합 His 태그 단백질의 정제 및 검출에 사용

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Ni-NTA Fast Start Kit (6)

Cat. No. / ID: 30600

6xHis-tagged 단백질 제제의 정제 및 검출에 사용: 6 x Fast Start Columns, Penta·His Antibody, 완충액, 시약

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₩610,000.00

Quantity

Ni-NTA Fast Start Kit은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

단백질 과학을 처음 접하는 연구자에게 적합
하나의 키트로 효율적인 정제에 필요한 모든 것을 제공
따라하기 쉬운 프로토콜 및 간단한 처리
단 90분 만에 컬럼당 단백질 최대 25mg 처리

Product Details

Ni-NTA Fast Start Kit는 미리 채워진 Ni-NTA 컬럼을 포함하여 투명한 E. coli 용해물에서 His 태그 단백질을 빠르고 효율적으로 정제하는 데 필요한 모든 것을 제공합니다. 키트에 제공된 완충액을 사용하면 기본 또는 변성 조건에서 단백질을 정제할 수 있습니다. 이 키트에는 발현된 His 태그 단백질을 검출하는 데 필요한 Anti-His 항체도 포함되어 있습니다.

Performance

Ni-NTA Fast Start Kit는 투명한 E. coli 용해물에서 His 태그 단백질을 빠르고 효율적으로 정제하는 데 필요한 모든 것을 제공합니다. 키트에 제공된 완충액을 사용하면 기본 또는 변성 조건에서 단백질을 정제할 수 있습니다(그림 참조). Ni-NTA Fast Start Kit는 단순하고 간단한 프로토콜과 즉시 사용 가능한 시약으로 인해 단백질 발현 분야로 연구를 확장하려고 하지만 단백질 정제 절차에 익숙하지 않은 과학자들이 사용하기 적합합니다.

See figures

기본 또는 변성 조건의 고순도 단백질

Principle

Ni-NTA Fast Start Kit는 6개 이상의 히스티딘 잔류물(연속 또는 교대)의 친화성 태그(His 태그)를 포함하는 단백질에 대한 특허받은 니켈-니트릴로트리아세트산(Nickel-Nitrilotriacetic Acid, Ni-NTA) 수지의 뛰어난 선택성을 기반으로 합니다. 이 기술을 사용하면 기본 또는 변성 조건에서 거의 모든 His 태그 단백질을 한 번에 정제할 수 있습니다. 니켈 이온에 대한 킬레이트화 부위가 4개인 NTA는 금속 이온과 상호 작용할 수 있는 부위가 3개뿐인 금속 킬레이트 정제 시스템보다 니켈을 더 단단히 결합합니다. 여분의 킬레이트화 부위는 니켈 이온 침출을 방지하여 다른 금속 킬레이트화 정제 시스템을 사용하여 얻은 것보다 순도가 더 높은 단백질 제제를 생성합니다. His 태그는 키트와 함께 제공되는 Penta·His Antibody에서 인식하는 epitope도 형성합니다.

Procedure

세포가 제공된 용해 완충액에 용해되고 원심분리되어 투명한 용해물이 생성됩니다. 이 용해물은 빠른 시작 컬럼에 적용되는데, 여기서 His 태그 단백질은 높은 친화성으로 결합되고 태그 없는 단백질은 통과합니다. 정제된 단백질은 세척 후 이미다졸을 포함(기본 조건)하거나 pH가 낮은(변성 조건) 완충액을 사용하여 용출됩니다(그림 참조). 정제 절차를 수행하고 단백질 수율을 얻은 후에는 제공된 Penta·His Antibody를 사용하여 SDS-PAGE, 웨스턴 블로팅, 면역검출을 수행할 수 있습니다.

See figures

고순도 His-tagged 단백질.

Applications

Ni-NTA Fast Start Kit는 6xHis 태그 단백질을 신뢰할 수 있는 단일 단계로 정제하여 다음을 포함한 모든 응용 분야에 적합합니다.

기능 연구
항체 생성을 위한 면역화
단백질-단백질 및 단백질-DNA 상호 작용을 포함하는 분석
구조 조사

Supporting data and figures

고순도 His-tagged 단백질.

Specifications

Features	Specifications
Applications	단백질체학
Support/matrix	Ni-NTA 기질
Processing	수동
Binding capacity	5~20mg/ml
Special feature	Ni-NTA 기술
Gravity flow or spin column	중력류
Start material	세포 용해물
Tag	6xHis tag
Yield	컬럼당 단백질 10mg 미만

Resources

FAQ

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

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

The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.

FAQ ID -836

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

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

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

The composition of the elution buffer in the Ni-NTA Fast Start Kit is 50 mM Na-phosphate, 300 mM NaCl and 250 mM imidazole at pH 8.0.

FAQ ID - 3353

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

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

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