The cell culture media containing HIV-1 particles were centrifuged at 1,200 × g for 5 min. The supernatants were passed through a 0.45-μm filter (ThermoFisher) and layered onto 20% sucrose for a 2-h ultracentrifugation at 25,000 × g at 4 °C. The virion pellets were subsequently isolated using anti-CD44 microbeads with a magnetic-based method according to the manufacturers’ instructions (µMACS™ VitalVirus HIV Isolation Kit, Miltenyi Biotec). The purified virions were lysed with RIPA buffer (ThermoFisher) and subjected to SDS-PAGE for western blotting as previously described (20 (link)).
Protocol detail
Isolation and purification of HIV-1 virions
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Buffer
Cd44
Cell
Cell culture
Culture media
Hiv 1
Isolation
Microbeads
Pellets
Ripa
Sds page
Sucrose
Ultracentrifugation
Virions
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Variable analysis
independent variables
- Centrifugation at 1,200 × g for 5 min
- Filtration through a 0.45-μm filter
- Ultracentrifugation at 25,000 × g for 2 h at 4 °C
- Isolation of virions using anti-CD44 microbeads with a magnetic-based method
- Presence and properties of HIV-1 particles in the final sample
- Temperature at 4 °C during ultracentrifugation
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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