Protocol for purifying recombinant HBV capsids from HepG2 cells and comparative phosphorylation analysis by phosphate-affinity SDS-PAGE
Key Points
To provide a practical protocol for purifying HBV capsids and analyzing their phosphorylation states for research purposes.
Transfected HepG2 cells to produce HBV capsids.
Used gradient ultracentrifugation for capsid purification.
Employed SDS-PAGE for phosphorylation profiling.
Analyzed phosphorylation states in capsid preparations.
Successfully purified HBV capsids from HepG2 cells.
Identified multiple phosphorylation sites on capsid proteins.
Established a protocol adaptable for various capsid preparations.
Abstract
The hepatitis B virus (HBV) capsid protein (HBc or Cp) contains multiple phosphorylation sites that regulate distinct stages of the viral lifecycle. Here, we present a protocol for purifying HBV capsids by gradient ultracentrifugation and analyzing their phosphorylation states. We describe detailed steps for transfection, gradient purification, capsid detection, concentration, and phosphorylation-level profiling. This protocol provides a practical approach to assessing the global phosphorylation state of purified HBV capsids and can be adapted for comparative analyses across different capsid preparations. For complete details on the use and execution of this protocol, please refer to Bianchini et al.1.