Recombinant

Recombinant Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)

Overview

  • Product name

    Anti-DNA:RNA hybrid antibody [S9.6]
  • Description

    Mouse monoclonal [S9.6] to DNA:RNA hybrid
  • Host species

    Mouse
  • Specificity

    The S9.6 monoclonal recognizes DNA-RNA hybrids (also known as R-loops) and does not bind to single or double stranded DNA (PubMed IDs: 2422282, 16614443). The antibody has high affinity for DNA-RNA hybrids but also binds RNA-RNA hybrids that are AU-rich (PubMed ID: 23784994). The specificity of the antibody appears to be determined by a combination of sequence and structural dependency since R-loop sequence affects binding affinity (PubMed ID: 28594954). It is important to use controls such as RNase A treatment in light of the cross-reactivity with double stranded RNA. Please see our DNA-RNA-IP (DRIP) protocol for further information. We do not recommend using this antibody for imaging applications such as ICC-IF, IHC or Flow due to the cross-reactivity with double stranded RNA. 

  • Tested applications

    Suitable for: IP, Dot blotmore details
    Unsuitable for: ICC/IF or IHC-P
  • Species reactivity

    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule. This information is considered to be commercially sensitive.

  • Positive control

    • DRIP: R-Loop. Dot blot: R-Loop.
  • General notes

    The pCALM3_2 vector used to generate R-loops was kindly provided by Prof. Frederic Chedin at UC Davis. The pCALM3_2 plasmid carries a 789bp fragment of the human CALM3 region that forms R-loops (PubMed ID: 31053798) cloned between T3 and T7 RNA polymerase promoter sequences. Transcription by T3 RNA polymerase leads to R-loop formation. Transcription by T7 RNA polymerase does not allow R-loop formation. 

    Our DNA-RNA immunoprecipitation (DRIP) protocol is linked here

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    S9.6
  • Isotype

    IgG2a
  • Light chain type

    kappa

Applications

Our Abpromise guarantee covers the use of ab234957 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP 1/200.

DNA-RNA hybrid Immunoprecipitation. Please find the DNA-RNA immunoprecipitation (DRIP) protocol link here.

Dot blot 1/1000.
  • Application notes
    Is unsuitable for ICC/IF or IHC-P.
  • Target

    • Relevance

      DNA-RNA hybrids occur naturally in eukaryotic cells, Particularly at sites of high transcriptional activity.

    Images

    • DNA-RNA hybrid Immunoprecipitation (DRIP) data using ab234957.

      M1 : 100bp DNA ladder                       

      M2 : High Range DNA Ladder 

      Lane 1: R-Loop ApaLI (RNase A treated)

      Lane 2: R-Loop (RNase A treated)

      Lane 3:R-Loop (RNase A+H treated)

      Lane 4: R-Loop ApaLI (RNase A+H treated)

      Lane 5: ab234957 with R-Loop ApaLI (RNase A treated)

      Lane 6: ab234957 with R-Loop(RNase A treated)

      Lane 7: ab234957 with R-Loop(RNase A+H treated)

      Lane 8: ab234957 with R-Loop ApaLI (RNase A+H treated)

      Lane 9-12: Flow through from IP

      Capture antibody: 5 μg

      Blocking buffer and concentration: 7.5 µg ssDNA.

      Diluting buffer and concentration: PBS containing 0.1% Triton X-100.

      A synthetic vector (pCALM3_2) was used to generate R-loops. ab234957 immunoprecipitates R-loops in the presence or absence of prior digestion by ApaLI, which does not affect R-loop structure. Prior treatment with RNase A (digests single stranded RNA) does not affect the IP signal whereas prior treatment with RNase H (digests RNA in DNA-RNA hybrids) eliminates the signal.

      The pCALM3_2 vector used to generate R-loops was kindly provided by Prof. Frederic Chedin at UC Davis.

    • Dot blot analysis with ab234957 at 1/1000 dilution.

      Lane 1: untranscribed plasmid.

      Lane 2: R-Loop.

      Lane 3: R-Loop (RNase A treated).

      Lane 4: R-Loop (RNase A+H treated).

      Lane 5: R-Loop (DNase I treated).

      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

      Blocking/Dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

      pCALM3_2 was cloned in pFC53 in the T7 orientation, but it forms R-loops transcribed in the reverse direction (that is with T3 promoter). DNase I has been shown to be less effective at digesting DNA in DNA-RNA hybrids compared to double stranded DNA (PubMed ID: 9020884). Prior treatment with DNase I reduced the signal by dot blot but did not eliminate it.

      The pCALM3_2 vector used to generate R-loops was kindly provided by Prof. Frederic Chedin at UC Davis.

    Protocols

    References

    This product has been referenced in:

    • Boguslawski SJ  et al. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J Immunol Methods 89:123-30 (1986). Read more (PubMed: 2422282) »
    See 1 Publication for this product

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    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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