Immunofluorescence using S9.6 antibody (Kerafast INC.) was performed as previously described (21 (link)). Cells were incubated with primary antibodies, anti-DNA-RNA hybrid [S9.6] antibody (1:500, Kerafast INC.), anti-nucleolin antibody (1:1000, Abcam Cat # ab22758), anti-PARP1 antibody (1:1000, Abcam Cat # ab32138 and 1:500 Cat # ab191217), anti- XRCC1 antibody (1:1000, Cell Signaling Technology Cat #27350), anti-DNA Polymerase beta antibody (1:1000, Abcam Cat # ab26343), anti-CYCLIN A antibody (1:200, Sigma Cat #C4710), and anti-RPA70/RPA1 antibody (1:50, Cell Signaling Technology Cat #2267S) followed by incubation in secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1000, Invitrogen Cat #A11034) or Alexa Fluor 594 goat anti-rabbit (1:1000, Invitrogen Cat #A11005). DNA was stained with DAPI. Images were captured at 40X magnification with a Zeiss Confocal Microscope (Zeiss LSM 700). Fiji software (version: 2.0.0-rc-69/1.52i, open source image processing software) used for analysis of images. As a control, cells were treated with 10 units of RNase H enzyme (New England Biolabs Cat # M0297L), prior to staining with S9.6 antibody.
Anti dna rna hybrid s9.6 antibody
Manufactured by Kerafast
The Anti-DNA-RNA hybrid [S9.6] antibody is a reagent used to detect and quantify DNA-RNA hybrid structures in biological samples. The antibody specifically recognizes and binds to DNA-RNA hybrid molecules, enabling their identification and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti parp1 antibody, by Abcam (2 mentions)
Anti xrcc1 antibody, by Cell Signaling Technology (2 mentions)
Anti cyclin a antibody, by Merck Group (2 mentions)
Anti rpa70 rpa1 antibody, by Cell Signaling Technology (2 mentions)
Anti dna polymerase beta antibody, by Abcam (2 mentions)
Rnase h enzyme, by New England Biolabs (2 mentions)
Anti nucleolin antibody, by Abcam (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
S9.6 antibody, by Kerafast (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Horseradish peroxidase conjugated goat specie specific secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Rnase h, by Merck Group (1 mentions)
5 protocols using anti dna rna hybrid s9.6 antibody
1
Immunofluorescence Analysis of DNA-RNA Hybrids
2
Dot Blot Analysis of DNA-RNA Hybrids
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Dot blot analysis was performed according to the protocol reported elsewhere (41 (link)). Genomic DNA was isolated by standard extraction with phenol/clorophorm/isoamylic alcohol (pH 8.0) followed by precipitation with 3 M NaOAc and 70% ethanol. Isolated gDNA was randomly fragmented overnight at 37°C with a cocktail of restriction enzymes (BsrgI, EcoRI, HindIII, XbaI) supplemented with 1 M Spermidin. After incubation, digested DNA was cleaned up with phenol/chloroform extraction and standard Ethanol precipitation. After sample quantification, 5 μg of digested DNA were incubated with RNase H overnight at 37°C as a negative control. Five micrograms of each sample were spotted onto a nitrocellulose membrane, blocked in 5% non-fat dry milk and incubated with the anti-DNA–RNA hybrid [S9.6] antibody (Kerafast) overnight at 4°C. Horseradish peroxidase-conjugated goat specie-specific secondary antibody (Santa Cruz Biotechnology, Inc.) was used. Quantification on scanned image of blot was performed using Image Lab software.
3
Immunofluorescence Quantification of DNA-RNA Hybrids
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Immunofluorescence using S9.6 antibody (Kerafast Inc.) was performed as previously described (21 (link)). Cells were incubated with primary antibodies, anti-DNA–RNA hybrid (S9.6) antibody (1:500, Kerafast Cat # ENH001), anti-nucleolin antibody (1:1,000, Abcam Cat # ab22758), anti-PARP1 antibody (1:1,000, Abcam Cat # ab32138 and 1:500 Cat # ab191217), anti-XRCC1 antibody (1:1,000, Cell Signaling Technology Cat #27350), anti-DNA Polymerase beta antibody (1:1,000, Abcam Cat # ab26343), anti-CYCLIN A antibody (1:200, Sigma Cat #C4710), and anti-RPA70/RPA1 antibody (1:50, Cell Signaling Technology Cat #2267S) followed by incubation in secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1,000, Invitrogen Cat #A11034) or Alexa Fluor 594 goat anti-rabbit (1:1,000, Invitrogen Cat #A11005). DNA was stained with DAPI. Images were captured at ×40 magnification with a Zeiss Confocal Microscope (Zeiss LSM 700). Fiji software (version: 2.0.0-rc-69/1.52i, open source image processing software) used for analysis of images. As a control, cells were treated with 10 U of RNase H enzyme (New England Biolabs Cat # M0297L), before staining with S9.6 antibody.
4
Detecting DNA-RNA Hybrids by DRIP
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The DRIP assays were modified from the previous study (Li et al., 2020a) . Tissues of interest were ground to fine powder in liquid nitrogen, and resuspended in genomic DNA extraction buffer (10-mM HEPES pH 7.5, 400-mM sucrose, 25-mM EDTA, 1-mM MgCl 2 , 0.5% [w/v] SDS, 1-mM PMSF, RNase inhibitor [SUPERase•In; Invitrogen]). DNA was recovered by phenol/chloroform extraction and then fragmented into pieces around 500 bp in size by sonication on ice. After DNA concentrations of different samples were measured by Nanodrop (Thermo Scientific), an equal amount of DNA for each sample was subjected to immunoprecipitation with the anti-DNA-RNA hybrid S9.6 antibody (Kerafast). RNase H (NEB) was added for negative controls during immunoprecipitation. The immunoprecipitation and subsequent qPCR procedures were followed as described in the ChIP assay.
5
DRIP Assay for DNA-RNA Hybrids
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DRIP assay was performed from activated CH12 cells using the anti-DNA-RNA hybrid (S9.6) antibody (Kerafast) based on the protocol in Ginno et al. (2012) .
Genomic DNA was digested with EcoRI, HindIII, NcoI, and BsOBI in the presence or absence of RNase H (Sigma) followed by phenol-chloroform extraction and ethanol precipitation. (Kerafast). The antibody against AID is previously published (Pavri et al., 2010) .
All primers used in this study are listed in Table S1.
Genomic DNA was digested with EcoRI, HindIII, NcoI, and BsOBI in the presence or absence of RNase H (Sigma) followed by phenol-chloroform extraction and ethanol precipitation. (Kerafast). The antibody against AID is previously published (Pavri et al., 2010) .
All primers used in this study are listed in Table S1.
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