Anti·His Antibodies, BSA-free

RGS·His 또는 HHHH 에피토프가 있는 His-tagged 단백질의 고감도 검출용

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RGS·His Antibody, BSA-free(100µg)

Cat. No. / ID:  34650

100µg mouse anti·RGS(H)4(동결 건조됨, BSA 무함유, 1000mL 작업 용액용)
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Tag
RGS·His
Tetra·His
Penta·His
Anti·His Antibodies, BSA-free은/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

  • C-말단, N-말단 및 내부 His tag의 높은 검출 특이성
  • RGS·His 에피토프의 고감도 고특이적 검출
  • HHHHH 에피토프의 고감도 고특이적 검출

Product Details

단일클론 마우스 Anti·His Antibody(5xHis 항체 및 4xHis 항체)는 RGS·His Antibody, BSA-free, Tetra·His Antibody, BSA-free 및 Penta·His Antibody, BSA-free와 같이 His tag를 운반하는 재조합 단백질을 검출하는 데 사용됩니다. 이 세 가지 변이체는 각기 다른 His tag 에피토프를 탐지합니다. 또한 모든 QIAGEN 마우스 단일클론 항체와 마찬가지로 바이러스, 마이코플라즈마 및 오염된 면역글로불린이 없는 무혈청 조건에서 제조됩니다.

Performance

QIAexpress Anti·His Antibody(5xHis 항체 및 4xHis 항체)를 사용하여 His tag를 통해 다양한 단백질이 검출되었습니다(표 'Anti∙His Antibody의 성능' 참조). 이 단백질 패널을 검출하는 세 가지 Anti·His Antibody의 민감도는  'QIAexpress Anti·His Antibody의 민감' 그림을 참조하십시오. Anti·His Antibody를 사용하면 콜로니 블로팅을 통해 양성 발현 클론을 식별하고(그림  '콜로니 블로팅을 통한 양성 발현 클론 식별' 참조), His-tagged 티오레독신을 검출할 수 있습니다(그림  '효모 세포에서 Penta·His Antibody에 의해 검출된 His-tagged 티오레독신' 참조). 이 항체는 또한 면역조직화학 절차에 사용하기에 이상적입니다(그림  'Penta·His Antibody를 사용한 면역조직화학' 참조).

Anti∙His Antibody의 성능

특성 RGS·His Antibody, BSA-free Tetra·His Antibody, BSA-free 및 Penta·His Antibody, BSA-free
검출 N-말단 His tag(에피토프 RGSHHHH 포함) N-말단, C-말단 및 내부 4xHis( Tetra·His Antibody, BSA-free) 또는 5xHis tag(Penta·His Antibody, BSA-free)
에피토프 검출됨 RGSHHHH HHHH(Tetra·His Antibody, BSA-free) 또는 HHHHH(Penta·His Antibody, BSA-free)
도트 블롯(dot blots)에서의 민감도* 0.5 ng 0.5 ng
웨스턴 블롯에서의 민감도* 2 ng 2 ng
아이소타이프(Isotype) 마우스 IgG1 마우스 IgG1
교차 반응성 천연 E. coli, 효모, 포유류 또는 곤충 세포 용해물에서는 무시할 수 있음 천연 E. coli, 효모, 포유류 또는 곤충 세포 용해물에서는 무시할 수 있음
형태 동결 건조됨 동결 건조됨
작업 용액 1000mL 1000mL

QIAexpress Anti∙His Antibody로 검출된 단백질

  단백질 태그 위치 염기서열
A DHFR N-말단 MRGSHHHHHHGSVRPLNCIV..........
B DHFR C-말단 .........VYEKKDGSRSHHHHHH
C 티오레독신 N-말단 MRGSHHHHHHGSSDKIIHLT..........
D TNF-α N-말단 MRHHHHHHQSTESMIRDV.........
E TFIIAγ N-말단 MGSSHHHHHHSSGLVPRGSH.....
F 샤페로닌 C-말단 ..........AKIAKDGSRSHHHHHH
G DNA 중합효소 C-말단 ..........EDWLSAKERSHHHHHH
H TFIIAαß 내부 ......QQQHQPQQQQHHHHHHHQQAQPQQTVP....

 

See figures

Principle

QIAexpress System은 6xHis-tagged 단백질의 발현, 정제, 검출 및 분석을 위한 재료를 제공합니다. 검출을 위해 QIAexpress 마우스 단일클론 항체는 최고의 순도와 활성을 보장하는 조건에서 생산됩니다. 하이브리도마 세포 배양은 무혈청 조건에서 수행되어 제제에 바이러스, 마이코플라즈마 및 오염된 면역글로불린이 없도록 보장합니다. 흡착 크로마토그래피에 의한 정제는 모두 생리적 pH에서 수행되므로 네이티브 항체 비율이 가장 높은 제제를 얻을 수 있습니다.

Procedure

모든 QIAexpress 검출 및 분석 제품과 함께 제공되는 QIAexpress, 검출 및 분석 안내서에는 분석 설계 및 수행을 위한 자세한 예시 프로토콜과 지침이 포함되어 있습니다.

Applications

RGS·His Antibody, BSA-free, Tetra·His Antibody, BSA-free 및 Penta·His Antibody, BSA-free를 포함한 QIAexpress Detection System은 His-tagged 단백질의 효율적인 검출을 가능하게 하며 다음을 포함한 많은 애플리케이션에 이상적입니다.

  • 콜로니, 도트 및 웨스턴 블로팅 절차
  • 양성 발현 클론 스크리닝
  • His-tagged 단백질의 발현 수준 및 안정성 모니터링
  • 면역 침전 및 ELISA
  • 면역세포화학 및 면역조직화학

Supporting data and figures

Resources

Kit Handbooks (1)
For Anti·His Antibodies, Anti·His HRP Conjugates, Penta·His™ Alexa Fluor® Conjugates, Penta·His Biotin Conjugate, Ni-NTA Conjugates, Tag·100™ Antibody, Streptavidin–R-PE, 6xHis Protein Ladder, Ni-NTA HisSorb™ Strips and Plates 
Supplementary Protocols (1)
Certificates of Analysis (1)

Publications

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.
Whitbeck JC; Connolly SA; Willis SH; Hou W; Krummenacher C; Ponce de Leon M; Lou H; Baribaud I; Eisenberg RJ; Cohen GH;
J Virol; 2001; 75 (1):171-80 2001 Jan PMID:11119586
Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.
Dierks T; Schmidt B; Borissenko LV; Peng J; Preusser A; Mariappan M; von Figura K;
Cell; 2003; 113 (4):435-44 2003 May 16 PMID:12757705
Screening for soluble expression of recombinant proteins in a 96-well format.
Knaust RK; Nordlund P;
Anal Biochem; 2001; 297 (1):79-85 2001 Oct 1 PMID:11567530
A human cDNA expression library in yeast enriched for open reading frames.
Holz C; Lueking A; Bovekamp L; Gutjahr C; Bolotina N; Lehrach H; Cahill DJ;
Genome Res; 2001; 11 (10):1730-5 2001 Oct PMID:11591650
Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments.
Dirac-Svejstrup AB; Shorter J; Waters MG; Warren G;
J Cell Biol; 2000; 150 (3):475-88 2000 Aug 7 PMID:10931861

FAQ

How should I dissolve the lyophilized Anti-His and Tag Antibodies?
Dissolve the lyophilized Anti-His Antibodies (100 µg) in 500 µl water per vial. The final concentration is 0.2 mg/ml. Dissolve the Anti-His Antibody Selector Kit antibodies (3 µg) in 15 µl water per tube (final concentration, 0.2 mg/ml).
-100
What epitopes do the Anti-His Conjugates and Anti-His Antibodies recognize?

The table below lists the epitopes recognized by the Anti-His Conjugates and Anti-His Antibodies. 'H' designates a histidine residue, while 'X' designates any amino acid other than histidine. Note that individual 6xHis-tagged proteins are often recognized better by one Anti-His Antibody than by the others, possibly because of subtle differences in the exact conformation of the 6xHis tag and other parts of the protein in the vicinity of the tag. To choose the optimal antibody for your specific 6xHis-tagged protein and application, QIAGEN offers the Anti-His Antibody Selector Kit. The kit includes 3 µg of each of the three antibodies for direct comparison and economical selection of the one that gives the best results.

Epitopes recognized by QIAexpress Anti-His HRP Conjugates and Anti-His Antibodies


Peptide Penta-His Tetra-His RGS-His
XHHHHHHX
+ +
XXHHHHHX
+ +
XHXHHHHX
+
XHHHHXHX
+
XRGSHHHHX
+ +
XRGSHHHHHHX
+ + +
XHXHHXHX

Note that the presence of specific residues N- or C-terminal to the epitope is not required.

FAQ ID -170
Which Anti-His Antibody is the most sensitive for my protein of interest?

Penta-His and Tetra-His Antibody bind to proteins that have 6xHis tags regardless of the surrounding amino-acids, even when the tag is partially hidden. Subtle differences in the antigen recognition sites of these antibodies mean that some 6xHis-tagged proteins are detected with higher sensitivity by one Anti-His Antibody than by the others (see figure below). Therefore, to achieve optimum results, we recommend comparison of these antibodies in parallel for new applications and proteins. QIAGEN offers the Anti-His Antibody Selector Kit for this purpose.


 

Sensitivity of QIAexpress Anti·His Antibodies



 

Note:  RGS His Antibody regonizes the RGS(His)4 epitope that is found on proteins endcoded by most pQE vectors as well as several pRSETan pBlue BacHis -vectors from Invitrogen.

FAQ ID -172
What are the dissociation constants for the Anti-His Antibodies?

Anti·His Antibody epitopes and dissociation constants

Antibody Epitope Dissociation constant Kd (M)*
RGS·His Antibody RGSHHHH 1 x 10–8 – 5 x 10–8
Penta·His Antibody HHHHH 5 x 10–8 – 1 x 10–9
Tetra·His Antibody HHHH 1 x 10–8 – 5 x 10–8

* Dissociation constants were measured using surface plasmon resonance (BIACORE) technology. The exact value of Kd is dependent on the individual 6xHis-tagged protein.

FAQ ID -385
Are QIAGEN monoclonal Anti His Antibodies glycosylated?
Yes, QIAGEN antibodies are glycosylated to a low extent. We did not determine whether this is N-glycosylation or O-glycosylation. Furthermore, we do not know, if the glycosylation pattern does change, since we do not measure it. Glycosylation does not have any impact onto the function of the antibody. If customers want to use the glycosylchains for coupling reactions we know that this is very difficult to do.
FAQ ID -334
How is an antibody epitope determined?
This is done via epitope mapping. The antibody in question is first tested against different protein fragments and peptides. Once the amino acids detected by the antibody are determined, the surrounding amino acids can be exchanged to reveal the influence of the environment on the detection of a specific epitope.
FAQ ID -593
What blocking reagents do you recommend for chemiluminescent detection using Penta-, Tetra-, and RGS-His antibodies?

Please do not use buffer containing milk powder to block the Western Blot. This will reduce sensitivity. QIAGEN recommends use 3% BSA in TBS-buffer to block the membrane and for incubation of the Anti His Antibody on the membrane. For the binding of the secondary Antibody BSA does not sufficiently block and therfore 10% milk powder in TBS-buffer should be used at this step only. Alternatively, if alkali-soluble casein (Merck, Cat. No. 1.02241) is available in your country it can be used as a blocking reagent throughout the chemiluminescent detection protocol. Please see Table 9 of the QIAxpress Detection and Assay Handbook for additional information.

FAQ ID -1093
What is the difference between BSA-free and BSA-containing RGS-His Antibody?

BSA stabilizes the RGS-His Antibody. We recommend to use the BSA-containing RGS-His Antibody for all standard procedures like dot blots, western blots, etc.. In cases where BSA may interfere with the application, e.g. coupling reactions, the RGS-His Antibody, BSA-free is recommended.

The Tetra-His and Penta-His Antibodies are stable without BSA, and therefore are only offered BSA-free.

FAQ ID -569
How should I store Anti·His and Tag·100 Antibodies?
QIAGEN Anti·His Antibodies should be stored lyophilized until they are to be used. They can be stored lyophilized for 1 year at 2–8°C. In solution they can be stored for 3 months at 2–8°C or for up to 6 months in aliquots at –20°C. Avoid repeated freezing and thawing.
FAQ ID -194