RNA-DNA Hybrid antibody

Details for Product No. ABIN4889499, Supplier: Log in to see
Antigen
Reactivity
All Species
Host
Mouse
Clonality (Clone)
Monoclonal ()
Application
Chromatin Immunoprecipitation (ChIP), Immunofluorescence (IF), Immunohistochemistry (IHC), In situ hybridization (ISH)
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'Independent Validation' Badge
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.

Primary Antibody ABIN4889499
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.

Validation Images
Surface Plasmon Resonance validation image for anti-RNA-DNA Hybrid antibody (ABIN4889499) The sensor response to the binding of RNA-DNA-hybrid antibody (left) and correspondin...
Immunogen DNA-RNA duplex (random RNA-DNA heteropolymer)
Clone D5H6
Isotype IgG
Specificity Reacts with RNA-DNA hybrid
Purification Purified (protein A)
Application Notes

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

Restrictions For Research Use only
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
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