pFastBac-GP67-H6HA1-His-RhPV-IRES-EGFP

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	The cells were then infected with vAcGP67-H6HA1-His-RhPV-IRES-EGFP baculovirus at an MOI of 1. The culture medium was collected by centrifugation after 72 h of infection. The protein in the culture medium was precipitated using ammonium sulfate fractionation at 80% saturation, and the pellet was resuspended in 10 mL phosphate buffered saline (PBS) buffer.

Protocol tips
The cells were then infected with vAcGP67-H6HA1-His-RhPV-IRES-EGFP baculovirus at an MOI of 1. The culture medium was collected by centrifugation after 72 h of infection. The protein in the culture medium was precipitated using ammonium sulfate fractionation at 80% saturation, and the pellet was resuspended in 10 mL phosphate buffered saline (PBS) buffer.

Publication protocol

Sf21 (2 × 107 cells) were seeded in a T75 flask and cultured at 28 °C in Sf-900II serum-free medium. The cells were then infected with vAcGP67-H6HA1-His-RhPV-IRES-EGFP baculovirus at an MOI of 1. The culture medium was collected by centrifugation after 72 h of infection. The protein in the culture medium was precipitated using ammonium sulfate fractionation at 80% saturation, and the pellet was resuspended in 10 mL phosphate buffered saline (PBS) buffer. After centrifugation, the clear supernatant was mixed completely with His-select nickel affinity gel (BIOMAN, Taiwan) overnight. The resin was then loaded into a column by gravity flow and was washed with 20 gel volumes of washing buffer (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol and 20 mM imidazole, pH 7.4). The recombinant H6HA1-His protein was then eluted with elution buffer (same as washing buffer except for the inclusion of 200 mM imidazole). All steps of protein purification were performed at 4 °C.

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