Anti·His Antibodies, BSA-free

Para detección sensible de proteínas con marcador His con un epítopo RGS·His o HHHH

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RGS·His Antibody, BSA-free (100 µg)

Cat. No. / ID:  34650

100 µg de anti-·RGS(H)4 de ratón (liofilizado, sin BSA, para solución de trabajo de 1000 ml)
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Tag
RGS·His
Tetra·His
Penta·His
Anti·His Antibodies, BSA-free están concebidos para su uso en aplicaciones de biología molecular. Estos productos no están concebidos para el diagnóstico, la prevención ni el tratamiento de enfermedades.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Detección altamente específica de etiquetas del extremo C, el extremo N y His internos
  • Detección extremadamente sensible y específica de epítopos RGS·His
  • Detección extremadamente sensible y específica de epítopos HHHHH

Product Details

Los anticuerpos monoclonales de ratón Anti·His (anticuerpos 5xHis y anticuerpos 4xHis), como RGS·His Antibody, BSA-free; Tetra·His Antibody, BSA-free; y Penta·His Antibody, BSA-free, se utilizan para detectar proteínas recombinantes portadoras de etiquetas His. Cada una de las tres variantes detecta un epítopo diferente del marcador His. Como todos los anticuerpos monoclonales de ratón de QIAGEN, se preparan sin suero, lo que garantiza la ausencia de virus, micoplasmas e inmunoglobulinas contaminantes.

Performance

Se han detectado muchas proteínas diferentes, a través de sus etiquetas His, usando QIAexpress Anti·His Antibodies (anticuerpos 5xHis y anticuerpos 4xHis) (consulte la tabla “Rendimiento de los Anti∙His Antibodies”). Para la sensibilidad de los tres anticuerpos Anti·His en la detección de este panel de proteínas, consulte la figura  “Sensibilidad de los QIAexpress Anti·His Antibodies”). Los Anti·His Antibodies posibilitan la identificación de clones de expresión positiva mediante la transferencia de colonias (consulte la figura  “Identificación de clones de expresión positiva mediante la transferencia de colonias”) y la detección de tiorredoxina con marcador His (consulte la figura  “Tiorredoxina con marcador His detectada mediante Penta·His Antibody en células de levaduras”). Los anticuerpos también son idóneos para su uso en procedimientos inmunohistoquímicos (consulte la figura  “Inmunohistoquímica con Penta·His Antibodies”).

Rendimiento de Anti∙His Antibodies

Característica RGS·His Antibody, BSA-free Penta·His Antibody, BSA-free y Penta·His Antibody, BSA-free
Detección Etiquetas His del extremo N (contienen el epítopo RGSHHHH) Etiquetas en el extremo N, el extremo C y 4xHis internos (Tetra·His Antibody, BSA-free) o 5xHis (Penta·His Antibody, BSA-free)
Epítopo detectado RGSHHHH HHHH (Tetra·His Antibody, BSA-free) o HHHHH (Penta·His Antibody, BSA-free)
Sensibilidad en dot blots* 0,5 ng 0,5 ng
Sensibilidad en Western blots* 2 ng 2 ng
Isotipo IgG1 de ratón IgG1 de ratón
Reactividad cruzada Insignificante con lisados celulares sin procesar de E. coli, levaduras, mamíferos o insectos Insignificante con lisados celulares sin procesar de E. coli, levaduras, mamíferos o insectos
Formulario Liofilizado Liofilizado
Solución de trabajo 1000 ml 1000 ml

Proteínas detectadas con QIAexpress Anti∙His Antibodies

  Proteína Ubicación del marcador Secuencia
A DHFR Extremo N MRGSHHHHHHGSVRPLNCIV..........
B DHFR Extremo C .........VYEKKDGSRSHHHHHH
C Tiorredoxina Extremo N MRGSHHHHHHGSSDKIIHLT..........
D FNT-α Extremo N MRHHHHHHQSTESMIRDV.........
E TFIIAγ Extremo N MGSSHHHHHHSSGLVPRGSH.....
F Chaperonina Extremo C ..........AKIAKDGSRSHHHHHH
G ADN polimerasa Extremo C ..........EDWLSAKERSHHHHHH
H TFIIAαß Interno ......QQQHQPQQQQHHHHHHHQQAQPQQTVP....

 

See figures

Principle

El QIAexpress System proporciona materiales para la expresión, purificación, detección y ensayo de proteínas con marcador 6xHis. Para la detección, se producen anticuerpos monoclonales de ratón QIAexpress en condiciones que garanticen la pureza y la actividad más elevada. El cultivo de células de hibridoma se lleva a cabo en condiciones libres de suero, lo que garantiza que las preparaciones estén libres de virus, micoplasmas e inmunoglobulinas contaminantes. La purificación por cromatografía de adsorción se lleva a cabo íntegramente en pH fisiológico, lo que permite obtener preparaciones con la mayor proporción de anticuerpo nativo.

Procedure

El Manual de uso de detección y ensayo de QIAexpress, suministrado con todos los productos de detección y ensayo QIAexpress, contiene directrices y protocolos de ejemplo detallados para diseñar y realizar ensayos.

Applications

Los QIAexpress Detection Systems, incluidos los anticuerpos RGS·His Antibody, BSA-free; Tetra·His Antibody, BSA-free; y Penta·His Antibody, BSA-free, permiten una detección eficiente de proteínas con marcador His y son ideales para muchas aplicaciones, como por ejemplo:

  • Procedimientos de transferencia de colonias, dot blots y Western blotting
  • Detección de clones de expresión positiva
  • Monitoreo de niveles de expresión y estabilidad de proteínas con marcador His
  • Inmunoprecipitación y ELISA
  • Inmunocitoquímica e inmunohistoquímica

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