Analysis of plasma protein adsorption onto polystyrene particles by two-dimensional electrophoresis: Comparison of sample application and isoelectricfocusing techniques

Two-dimensional electrophoresis (2-DE) was previously established for analy sis of plasma protein adsorption patterns on particulate carriers for intra venous drug targeting. This study addresses a possible effect of polymeric particles on protein separation in the first dimension, e.g., hindrance of protein entry into the gel or interaction of particles with the gel matrix. Polystyrene beads of mean diameter 100, 200 and 1000 nm were used as model carriers. Two different separation techniques were performed in the first dimension of 2-DE to study possible interactions of the beads with the diff erent gel matrices, i.e., carrier ampholytes (CA) and immobilized pH gradie nts (IPGs). Comparison of gels obtained from samples including the particle s from samples separated from the polystyrene beads showed no noteworthy di fferences. Therefore, a negative effect of the particles can be excluded, a nd particle separation from the sample is not necessary. Another goal of th is study was the transfer of analytical protocols for isoelectric focusing from CA to IPGs with regard to enhanced reproducibility, faster sample proc essing, and easier handling. Transfer from CA to IPGs was carried out succe ssfully and showed improved resolution of basic proteins. In contrast to th at, lower amounts of a few high molecular mass proteins were detected, espe cially when sample application cups were employed. A qualitative change in the obtained protein pattern was not observed. Increased entry of high mole cular weight proteins was achieved by in-sample rehydration instead of usin g sample cups.