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Figure 7 | Mobile DNA

Figure 7

From: Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

Figure 7

Monoclonal anti-ORF2p antibody inhibits L1 endonuclease activity in an in vitro endonuclease cleavage assay. (A) Schematic of in vitro endonuclease cleavage assay. Double-stranded DNA containing L1 ORF2 endonuclease consensus target sequence with 5ā€² tagged with fluorophore. L1 ORF2 endonuclease is added, DNA is cleaved releasing the fluorphore, which can be quantitated. (B) (Top) SDS-PAGE analysis of the products resulting from the in vitro endonuclease assay with or without the addition of the monoclonal anti-ORF2 antibody (0, 100, 150, 200 nM). Antibody (ORF2) denotes the addition of the monoclonal anti-ORF2 antibody, control indicates the addition of the same volume of the buffer used for the reactions containing monoclonal anti-ORF2p antibody, and L1 EN denotes bacterially purified human ORF2 endonuclease. (Bottom) Quantitation of the results of the in vitro endonuclease cleavage assay in A (see Methods). Results were normalized to 0 nM control (nā€‰=ā€‰3). (C) Same experimental approach as in B, but anti-hORF1p antibody was added to the in vitro endonuclease cleavage assay.

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