Immunoprecipitation of Nipah Virus Matrix Protein

293T cells in 6-wells were transfected with 1 ug of expression constructs or empty pcDNA3 vector for a total of 2 ug per well, using BioT transfection reagent (Bioland) according to manufacturer protocol. At 24 hours post-transfection, cells were lysed in cold lysis buffer (1% NP-40, 100 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1X protease inhibitor cocktail (Roche)), clarified at 14k rpm for 5 minutes at 4°C, and incubated with 3 ug of rabbit anti-NiV-M [50 (link)] or rabbit anti-HA (Novus, NB600-363) at 4°C overnight. Following incubation with protein G agarose (Pierce) for 2 hours at 4°C, the bound protein was washed 4 times with cold wash buffer (same composition as lysis buffer but with 0.2% NP-40), then eluted in reducing Laemmli SDS sample buffer at 95°C.