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

• IP

Supplier Data

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.

All lanes:

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

false

• Dot

Supplier Data

Dot Blot - Anti-DNA:RNA hybrid antibody [S9.6] (AB234957)

Dot blot analysis of DNA : RNA hybrid using ab234957 at 1/1000 dilution followed by a Goat Anti-Mouse IgG (H+L) at 1/5000 dilution.

Blocking and diluting buffer : 5% NFDM/TBST.

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.

pFC53 is used to generate R-loops.

All lanes:

Dot Blot - Anti-DNA:RNA hybrid antibody [S9.6] (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)

Secondary

All lanes:

Goat Anti-Mouse IgG (H+L) at 1/5000 dilution

false

Exposure time: 1s