**2.3 Nanoparticle/plasma incubation**

Nanoparticles were dissolved (final concentration 2 mg/ml) in PBS and incubated with 1% plasma at 37C for 1h. As control, to ensure there was no protein precipitation, one sample was prepared without nanoparticles. Unbound proteins were separated from nanoparticles by centrifugation for 40 min at 50 000g and 4°C. The supernatant was discarded and the particle pellet was washed in Dithiothreitol (DTT, Sigma-Aldrich) 20mM/-acetone buffer followed by a second centrifugation step for 10 min at 50 000g and 4°C. The supernatant was discarded and the pellet was air dried. The pellets containing nanoparticles and attached proteins were then dissolved in denaturing solution containing 9M Urea (Sigma-Aldrich), 65mM DTT and 4% (3-(3-cholamidopropyl)-dimethylamino)-1-propanesulfonate (CHAPS). The solution was incubated at room temperature for 30 min before denatured proteins were separated from the nanoparticles by centrifugation for 30 min at 50 000g and 4°C. The supernatant was collected and the samples were analyzed in triplicates. 20 µl (40µg protein) was applied on the IEF strip in each 2-DE analysis.
