Anti·His Antibodies, BSA-free

Zum sensitiven Nachweis von Proteinen mit His-tag mit einem RGS·His- oder HHHH-Epitop

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RGS·His Antibody, BSA-free (100 µg)

Cat. No. / ID:  34650

100 µg Maus-Anti-RGS(H)4 (lyophilisiert, BSA-frei, für 1000 ml Arbeitslösung)
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744,00 €
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Tag
RGS·His
Tetra·His
Penta·His
Anti·His Antibodies, BSA-free sind für molekularbiologische Anwendungen vorgesehen. Diese Produkte sind nicht zur Diagnose, Prävention oder Behandlung einer Erkrankung vorgesehen.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Hochspezifischer Nachweis von C-terminalen, N-terminalen und internen His-tags
  • Hochsensitiver und spezifischer Nachweis von RGS·His-Epitopen
  • Hochsensitiver und spezifischer Nachweis von HHHHH-Epitopen

Product Details

Zum Nachweis rekombinanter Proteine mit His-tag kommen monoklonale Maus-Anti·His-Antikörper (5xHis-Antikörper und 4xHis-Antikörper) wie der BSA-freie RGS·His-Antikörper, der BSA-freie Tetra·His-Antikörper und der BSA-freie Penta·His-Antikörper zum Einsatz. Jede der drei Varianten weist ein anderes His-tag-Epitop nach. Wie alle monoklonalen Maus-Antikörper von QIAGEN werden sie unter serumfreien Bedingungen hergestellt, was gewährleistet, dass sie keine Viren, Mykoplasmen und kontaminierenden Immunglobuline enthalten.

Performance

Mit QIAexpress Anti·His-Antikörpern (5xHis-Antikörper und 4xHis-Antikörper) wurden viele verschiedene Proteine über ihre His-tags nachgewiesen (siehe Tabelle “Leistung der Anti∙His-Antikörper”). Die Sensitivität der drei Anti·His-Antikörper beim Nachweis dieser Gruppe von Proteinen ist in der Abbildung  “Sensitivität der QIAexpress Anti·His-Antikörper” dargestellt. Anti·His-Antikörper ermöglichen die Identifizierung positiver Expressionsklone durch Kolonie-Blotting (siehe Abbildung  “Identifizierung positiver Expressionsklone durch Kolonie-Blotting”) und den Nachweis von Thioredoxin mit His-tag (siehe Abbildung  “Nachweis von Thioredoxin mit His-tag durch Penta·His-Antikörper in Hefezellen”). Die Antikörper eignen sich auch hervorragend für den Einsatz in der Immunhistochemie (siehe Abbildung  “Immunhistochemie mit Penta·His-Antikörpern”).

Leistung der Anti∙His-Antikörper

Eigenschaften RGS·His Antibody, BSA-free Tetra·His Antibody, BSA-free und Penta·His Antibody, BSA-free
Nachweis N-terminale His-tags (die das Epitop RGSHHHH enthalten) N-terminale, C-terminale und interne 4xHis-tags (Tetra·His Antibody, BSA-free) oder 5xHis-tags (Penta·His Antibody, BSA-free)
Epitopnachweis RGSHHHH HHHH (Tetra·His Antibody, BSA-free) oder HHHHH (Penta·His Antibody, BSA-free)
Sensitivität bei Dot-Blots* 0,5 ng 0,5 ng
Sensitivität bei Western-Blots* 2 ng 2 ng
Isotyp Maus-IgG1 Maus-IgG1
Kreuzreaktivität Vernachlässigbar bei E. coli-Rohlysaten, Hefe-, Säuger- oder Insektenzelllysaten Vernachlässigbar bei E. coli-Rohlysaten, Hefe-, Säuger- oder Insektenzelllysaten
Form Lyophilisiert Lyophilisiert
Arbeitslösung 1000 ml 1000 ml

Mit QIAexpress Anti∙His-Antikörpern nachgewiesene Proteine

  Protein Tag-Lokalisation Sequenz
A DHFR N-Terminus MRGSHHHHHHGSVRPLNCIV..........
B DHFR C-Terminus .........VYEKKDGSRSHHHHHH
C Thioredoxin N-Terminus MRGSHHHHHHGSSDKIIHLT..........
D TNF-α N-Terminus MRHHHHHHQSTESMIRDV.........
E TFIIAγ N-Terminus MGSSHHHHHHSSGLVPRGSH.....
F Chaperonin C-Terminus ..........AKIAKDGSRSHHHHHH
G DNA-Polymerase C-Terminus ..........EDWLSAKERSHHHHHH
H TFIIAαß Intern ......QQQHQPQQQQHHHHHHHQQAQPQQTVP....

 

See figures

Principle

Das QIAexpress System bietet Materialien für Expression, Aufreinigung, Nachweis und Assays von Proteinen mit 6xHis-tag. Für den Nachweis werden monoklonale QIAexpress-Maus-Antikörper unter Bedingungen hergestellt, die höchste Reinheit und Aktivität sicherstellen. Die Hybridomzellkultur wird unter serumfreien Bedingungen durchgeführt, sodass die Präparationen frei von Viren, Mykoplasmen und kontaminierenden Immunglobulinen sind. Die Aufreinigung durch Adsorptionschromatographie wird ausschließlich bei physiologischem pH-Wert durchgeführt, sodass Präparationen mit dem höchsten Anteil an nativen Antikörpern entstehen.

Procedure

Das QIAexpress Nachweis- und Assayhandbuch, das mit allen QIAexpress Nachweis- und Assayprodukten geliefert wird, enthält detaillierte Beispielprotokolle und Leitfäden für die Entwicklung und Durchführung von Assays.

Applications

QIAexpress Nachweissysteme, einschließlich RGS·His Antibody, BSA-free, Tetra·His Antibody, BSA-free und Penta·His Antibody, BSA-free, ermöglichen den effizienten Nachweis von Proteinen mit His-tag und sind ideal für viele Anwendungen wie:

  • Kolonie-, Dot- und Western-Blotting-Verfahren
  • Screening auf positive Expressionsklone
  • Monitoring von Expressionsniveau und Stabilität von Proteinen mit His-tag
  • Immunopräzipitation und ELISA
  • Immunozytochemie und Immunhistochemie

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