RNA-DNA Hybrid Antikörper

Produktnummer ABIN4889499

Produktdetails

Target: RNA-DNA Hybrid

Reaktivität: Alle Spezies

Wirt: Maus

Klonalität: Monoklonal

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

Spezifität: Reacts with RNA-DNA hybrid

Aufreinigung: Purified (protein A)

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

Klon: D5H6

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