| Assay Method Information | |
| | EN PA FRET Inhibition Assay |
| Description: | EN PA FRET inhibition assay was performed using a 19 nucleotide synthetic oligoribonucleotide substrate: 5′-FAM-AUUUUGUUUUUAAUAUUUC-BHQ-3′ (Integrated DNA Technologies, Inc., Coralville, Iowa) (SEQ. ID. NO. 1). Upon RNA cleavage, the fluorescent FAM group is released from the BHQ quencher. The PA sequence used to produce active enzyme is derived from any one of multiple influenza A virus strains (e.g., A/goose/Nanchang/3-120/01 (H3N2), A/Victoria/3/1975 (H3N2), A/Brisbane/10/2007 (H3N2), A/WSN/33 (H1N1), A/CA/4/2009 (H1N1), A/CA/5/2009 (H1N1), A/Shanghai/1/2013 (H7N9), A/Guizhou/1/2009 (H5N1)). The full length recombinant protein was expressed from a baculovirus vector in insect cells. Full length EN PA was used in this assay at an effective concentration of 1 to 10 nM, together with 50 nM FRET probe with a final volume of 20 ml cleavage buffer (20 mM Tris Ph8, 100 mM NaCl, 5% Glycerol, 10 mM 3-ME, 0.0003% Tween-20, 5 mM MgC2).Compounds described herein were added to a 384-well black polypropylene plate. Fluorescence was measured in a continuous mode up to 120 minutes with a Wallac 1420 Victor3V multilabel counter (PerkinElmer Life Sciences, Shelton, Conn.) (excitation 485 nm; emission 535 nm). |
| Affinity data for this assay | |
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