RNA-DNA Hybrid antibody

Catalog No. ABIN4889499

Product Details

Target: RNA-DNA Hybrid

Reactivity: All Species

Host: Mouse

Clonality: Monoclonal

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

Specificity: Reacts with RNA-DNA hybrid

Purification: Purified (protein A)

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

Clone: D5H6

Isotype: IgG

Application Details

Application Notes:

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

Restrictions: For Research Use only

Independent Validation (SPR)

Validation #100000 (Surface Plasmon Resonance)

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

No.: #100000

Date: 06/23/2015

Antigen: RNA-DNA Hybrid

Method validated: Surface Plasmon Resonance

Positive Control: RNA-DNA Hybrid complexes

Negative Control: ssDNA, dsDNA

Notes:

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

Validation Images

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

Full Methods

Primary Antibody: 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.

Experimental Notes:

• 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.

Handling

Format: Lyophilized

Reconstitution: Must be reconstituted in distilled water.

Concentration: 1 mg/mL

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

Storage: 4 °C/-20 °C

Storage Comment: 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.

Expiry Date: 24 months

Target Details

Target: RNA-DNA Hybrid