CST, 47099T, DNA-RNA Hybrid (S9.6) Mouse Monoclonal Antibody

Monoclonal Antibody for studying . Validated for Functional. Available in 2 sizes. Highly specific and rigorously validated in-house, DNA-RNA Hybrid (S9.6) Mouse Monoclonal Antibody (CST #47099) is ready to ship. Product Usage Information Dot-blot analysis of R-loops using hybridized DNA-RNA, double-stranded DNA (dsDNA), and double-stranded RNA (dsRNA) species performed according to the detailed protocol published in PMID: 33554969. Briefly: 1. Order oligonucleotides based on PMID: 33554969. Resuspend in annealing buffer at 10 mM Tris (pH 8.0), 50 mM NaCl, and 1 mM EDTA to reach a final concentration of 100 µM. 2. Generate 10 mM hybridized DNA-RNA, dsDNA, and dsRNA species by incubating equimolar amounts of the corresponding oligonucleotides in annealing buffer, heating them up to 95°C for 10 min, and then allowing them to cool slowly to room temperature to allow for reannealing of the strands. 3. Prepare serial dilutions of hybridized oligos at 80, 20, 10, and 4 nM final concentrations prepared in annealing buffer, load 50 mL of each sample into separate wells of the 96-well dot-blot apparatus, then apply to a pre-wet nylon membrane. 4. Apply gentle vacuum pressure to draw solution through the membrane and wrap the mostly dry membrane in plastic wrap. 5. UV cross-link the membrane at 1,200 J/m2 twice using a UV crosslinker. 6. Prepare blocking buffer: 5% milk in Tris-buffered saline with 0.05% Tween 20 (TBST); incubate the membrane in 25 mL of blocking buffer with gentle agitation for 1 hr at room temperature. 7. Wash the membrane three times for 5 min each with 15 mL of 1X TBST, then incubate overnight in primary antibody at 1:1,000 dilution in 10 mL of blocking buffer with gentle agitation at 4°C. 8. Wash the membrane three times for 5 min each with 15 mL of 1X TBST, then incubate with Anti-mouse IgG, HRP-linked Antibody # 7076 at 1:2,000 in 10 mL of blocking buffer with gentle agitation for 1 hr at room temperature. 9. Wash the membrane three times for 5 min each with 15 mL of 1X TBST, then develop with enhanced chemiluminescence (ECL) reagents to acquire signals for imaging. Functional: 1:1000 Storage Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at -20°C. Do not aliquot the antibody. Specificity / Sensitivity DNA-RNA Hybrid (S9.6) Mouse Monoclonal Antibody recognizes endogenous levels of DNA-RNA R-loops. This antibody has been validated using dot-blot assays and shows specificity for Species Reactivity: All Species Expected Source / Purification Monoclonal antibody is produced by immunizing animals with the DNA-RNA heteropolymer duplex generated by transcription of φX174 single-stranded DNA (ssDNA) with DNA-dependent RNA polymerase. Background R-loops are three-stranded nucleic acid structures composed of a DNA-RNA hybrid and a single-stranded DNA (ssDNA). These structures form during transcription when a newly synthesized nascent RNA basepairs with its DNA template strand and displaces the non-template DNA strand. R-loops are recognized as playing key roles in various cellular processes, including gene regulation, transcription termination, and DNA damage response (1). Although R-loops form naturally during transcription, their persistent formation and lack of proper resolution can lead to deleterious effects on genome integrity (2). Related to this, aberrant R-loop formation and accumulation have been linked to a growing number of human diseases, including neurodegenerative conditions, Friedreich Ataxia and Amyotrophic Lateral Sclerosis (ALS), intestinal inflammation, and most significantly, various cancers (3-6). Specification REACTIVITY: All SENSITIVITY: Endogenous Source/Isotype: Mouse IgG2a