This H1N1 Nucleoprotein/NP overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Nucleoprotein/NP protein (Cat: 11675-V08B) from the overexpression lysate was verified.
A DNA sequence encoding the Influenza A virus (A/Puerto Rico/8/34/Mount Sinai (H1N1)) nucleoprotein (AAM75159.1, with mutation Ile 116 Met) (Met1-Gly490) was fused with a polyhistidine tag at the C-terminus.
The recombinant Influenza A virus (A/Puerto Rico/8/34/Mount Sinai (H1N1)) nucleoprotein comprises 501 amino acids and has a predicted molecular mass of 56.7 kDa. It migrates as an approximately 50 kDa band in SDS-PAGE under reducing conditions.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Influenza A viral nucleoprotein (NP) plays a critical role in virus replication and host adaptation. The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. The nucleocapsid is a complex of the viral nucleoprotein, RNA, and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins.