Fluorescent Functionalization of Virus-Like Particles via Disulfide Re-bridging

Begin with virus-like particles, or VLPs, containing disulfide bonds exposed on the capsid surface.

Add a reducing agent to cleave the disulfide bonds, generating free thiol groups while preserving the capsid structure.

Combine the reduced VLPs with a thiol-reactive polyethylene glycol, or PEG linker, to prevent thiol reoxidation.

The PEG linker re-bridges each thiol pair by forming two covalent bonds.

This covalent conjugation also forms a fluorescent structure that appears bright yellow under UV illumination.

Rotate the mixture for an extended period to allow full conjugation.

Using centrifugal filtration, purify the PEGylated VLPs by removing unreacted small molecules while retaining intact conjugates.

Perform non-reducing SDS-PAGE to detect PEG attachment, indicated by a fluorescent coat protein band.

Assess structural integrity using native agarose gel electrophoresis, where similar bands for PEGylated and non-PEGylated VLPs confirm the presence of intact VLPs.

This protocol yields fluorescent, PEG-functionalized VLPs suitable for imaging or targeted delivery applications.

This schematic shows the conjugation that will be demonstrated. 10 equivalents of Tris(2-carboxyethyl)phosphine or