This is the protocol for fluorescent labeling of purified SV40 particles as originally developed by Lucas Pelkmans. The resulting labeled virus particles can be used in timelapse imaging and single-particle tracking. This protocol can be up- or down-scaled depending on the amount of purified virus one wishes to label, and can be adapted to couple all kinds of different labels to the virus particles (e.g. biotin).
- Take 1 mg of purified virus at 1 mg/ml in 0.1 M carbonate buffer pH 8.3. Incubate this with 33 μl of fluorophore-X-succinimidyl ester (10 mg/ml in DMSO) for 1 h at RT by end-over-end rotation in an eppendorf tube wrapped in aluminum foil to prevent light exposure. The fluorophore can be different, such as Texas Red, Fluorescein, or different forms of Alexa Fluor (Molecular Probes/Invitrogen). These fluorophores react exclusively with free amines, resulting in a stable carboxamide bond and contain a seven-atom aminohexanoyl spacer (X), which allows higher degrees of labeling without functional perturbance of the virus.
- After labeling, the virus can be either re-purified using CsCl and subsequent dialysis (see purification protocol), or be separated from unreacted fluorophore using desalting columns such as the PD-10 or PD MiniTrap G25 (GE Healthcare). Pre-wash the column and elute the virus from the column with virion buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM CaCl2). Note that the virus elutes first from the column, and will eventually be diluted about twice. Conveniently, unreacted fluorophore will react with the column material, thus staying behind in the column.
- The collected virus should be stored at –20ºC (using 10% Glycerol to prevent freezing) in 2 μg aliquots.