Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)
![Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)](https://www.abcam.com/ps/products/234/ab234957/Images/ab234957-350851-mouse-monoclonal-s96-to-dna-rna-hybrid-drip-rloop.jpg "Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Mouse monoclonal [S9.6] to DNA:RNA hybrid
- Suitable for: IP, Dot blot
- Reacts with: Species independent
Overview
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Product name
Anti-DNA:RNA hybrid antibody [S9.6]
See all DNA:RNA hybrid primary antibodies -
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.
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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.
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Positive control
- DRIP: R-Loop. Dot blot: R-Loop.
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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
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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
pH: 7.2
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
Images
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Immunoprecipitation - Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)
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.
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![Dot Blot - Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)](https://www.abcam.com/ps/products/234/ab234957/Images/ab234957-350852-mouse-monoclonal-s96-to-dna-rna-hybrid-dot-blot.jpg)
Dot Blot - Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)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.
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![Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)](https://www.abcam.com/ps/products/234/ab234957/Images/ab234957-4-benefits-of-recombinant-antibodies.png)
Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)
