Affinity Purification of Influenza Viral Ribonucleoproteins

NP, PB2, FLAG-tagged PB1, FLAG-tagged PA, and segment 6 vRNA (NA) were expressed in transfected HEK 293T cells for 48 hr, following prior approaches [54 (link)]. Cells were lysed in co-IP buffer in the presence of protease inhibitors. Lysates were clarified by centrifugation, pre-cleared protein A agarose (Santa Cruz Biotech sc-2001) for 1 hr, and transferred to a new tube where BSA was added to a final concentration of 5 mg/mL. NP was immunoprecipitated overnight with 3 μg anti-NP antibody. Immunocomplexes were captured using protein A agarose (Sigma P2545) for 1 hr, washed twice with co-IP buffer containing 5 mg/mL BSA and 500 mM NaCl, and twice with co-IP buffer. Bound proteins were eluted by boiling in Laemmli buffer. Samples were then assayed via western blot analysis for presence of NP, PB1, and PA.