DNA-RNA Hybrid (S9.6) Mouse Monoclonal Antibody #47099

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.