RNA-DNA Hybrid Antikörper

Produktnummer ABIN4889499

Dieser Anti- Antikörper ist ein Maus Monoklonal Antikörper zur Detektion von in ChIP, IF, IHC und ISH. Geeignet für Alle Spezies. Unabhängig validiert für die Verwendung in Surface Plasmon Resonance.

Kurzübersicht für RNA-DNA Hybrid Antikörper (ABIN4889499)

Target: RNA-DNA Hybrid

Reaktivität: Alle Spezies

Wirt: Maus

Klonalität: Monoklonal

Applikation: Chromatin Immunoprecipitation (ChIP), Immunofluorescence (IF), Immunohistochemistry (IHC), In situ hybridization (ISH)

Klon: D5H6

Produktdetails

Spezifität: Reacts with RNA-DNA hybrid

Aufreinigung: Purified (protein A)

Immunogen: DNA-RNA duplex (random RNA-DNA heteropolymer)

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise:

Working dilution: Optimal dilutions should be determined by the end user.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (SPR)

Validierung #100000 (Surface Plasmon Resonance)

by: Institute of Photonics and Electronics AS CR, v.v.i.

No.: #100000

Datum: 23.06.2015

Antigen: RNA-DNA Hybrid

Validierte Anwendung: Surface Plasmon Resonance

Positivkontrolle: RNA-DNA Hybrid complexes

Negativkontrolle: ssDNA, dsDNA

Bewertung:

The ability of the D5H6 antibody to recognize and bind RNA/DNA hetero-duplexes was approximately twice lower than expected.

Validierungsbilder

The sensor response to the binding of RNA-DNA-hybrid antibody (left) and corresponding calibration curve (right).

Protokoll

Primärantikörper: ABIN4889499

Full Protocol:

• For the experiments a four-channel spectroscopic SPR sensor with a temperature controller developed at IPE was used. In general, the SPR assay was based on the attachment of biotinylated DNA probes to the SPR sensor surface via the streptavidin-biotin interaction, with the streptavidin covalently attached to the alkanthiol self-assembled monolayer, subsequent forming of RNA/DNA hybrid duplexes and monitoring of their interaction with RNA-DNA-hybrid antibody.

• The respective steps of the assay were as follows:

• 1. Streptavidin was covalently attached to the sensor surface via amine coupling according to the previously published protocol (Vaisocherová et al. (2006) Biopolymers 82:394-398).
  2. Biotinylated DNA probes were immobilized in 10mM Tris buffer. The amount of probes was calculated to be of 10¹² probes/cm² and was kept the same across all experiments.
  3. Complementary miRNA strands were bound in 10mM Tris + 15mM MgCl₂ to form RNA-DNA hybrid duplexes. The concentration range of miRNA used was 0.1-100nM.
  4. The D5H6 at a concentration of 1µg/ml was used in all experiments.

• All the experiments were performed at the temperature of 25°C and the flow rate of 20µl/ml.

Anmerkungen:

• The sensor response to the binding of D5H6 antibody obtained for various concentrations of miRNA and corresponding calibration curve are shown at Fig.1. The calibration curve is plotted as a dependence of the sensor response to the binding of D5H6 antibody on the concentration of miRNA and is linear up to the miRNA concentration of 5nM.

• As a negative control, the ssDNA (only biotinylated DNA probe) or dsDNA (homo-duplex with DNA analogue of the miRNA) surface was used. In both cases, the nonspecific sensor response to the binding of D5H6 was below 2% which falls into the inter-assay variability range.

Handhabung

Format: Lyophilized

Rekonstitution: Must be reconstituted in distilled water.

Konzentration: 1 mg/mL

Buffer: Tris 0,1M, glycine 0,1M, sucrose 2 %

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Lyophilized powder stable for a minimum of 2 years at -20°C. Store reconstituted antibodies at +4°C. For extended periods store in aliquots at -20°C. Antibodies are guaranteed for 6 month from date of receipt.

Haltbarkeit: 24 months

Antigendetails

Target: RNA-DNA Hybrid

Substanzklasse: Nucleotide