Fig. 2.

Characterization of fluorescence molecular painting (FMP): a molecular painting is dependent on lipid residues in the GPI anchor. MonoGGhis protein containing an intact GPI-anchor was used for FMP, as well as protein pre-treated with PI-PLC, thus rendering it hydrophilic. Comparable amounts of LV virus particles and GPI-AP were used for the experiments. The signal in V+ is lost, when PI-PLC was used to pre-treat the protein. p24 immunoblots indicate the levels of viral particles present. P is protein controls for loading of protein and gauging comparable amounts of protein in the test lanes based on the respective binding of antibodies. b Duplex painting. CD59his and monoGGhis were used simultaneously to modify LV particles. No signals were observed for GFP and CD59 in the M+ and V− samples. In the V+ CD59his sample, a signal was only visible in the CD59-specific blot, but not the GFP-specific detection, and vice versa for the V + GFP samples, indicating successful single MP. In V++ signals were detected with both antibodies. No significant difference is seen between signal strength in single and double painted samples. p24 immunoblots indicate the levels of viral particles present. P is protein controls for loading of protein and gauging comparable amounts of protein in the test lanes based on the respective binding of antibodies. M+ medium incubated with GPI-AP during FMP, V− virus suspension incubated in the absence of GPI-AP during FMP, V+ virus suspension incubated with GPI-AP during FMP, P purified GPI-AP/p24 control