Immunoblot analysis of proteins associated with self-assembled monolayer surfaces of defined chemistries.

Abstract

Intact and fragmented proteins, eluted from self-assembled monolayer (SAM) surfaces of alkanethiols of different chemistries (-CH₃, -OH, -COOH, -NH₂), following exposure to human plasma (HP) or human serum (HS), were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The SAM surfaces were incubated for 1 h with 10% (v/v) sterile-filtered, heat-inactivated (h.i.) HS or 1% (v/v) sterile-filtered h.i. HP preparations [both in phosphate buffered saline (PBS)]. Adsorbed proteins were eluted using 10% SDS/2.3% dithioerythritol for characterization of protein profiles. The type of incubating medium may be an important determinant of adsorbed protein profiles, since some variations were observed in eluates from filtered versus control unfiltered h.i. 10% HS or 1% HP. Albumin and apolipoprotein A1 were consistently detected in both filtered h.i 10% HS and 1% HP eluates from all SAM surfaces and from control tissue culture-treated polystyrene (TCPS). Interestingly, Factor H and Factor I, antithrombin, prothrombin, high molecular weight kininogen (HMWK), and IgG were present in eluates from OH, COOH, and NH₂ SAM surfaces and in eluates from TCPS but not in eluates from CH₃ SAM surfaces, following exposure to filtered h.i. 10% HS. These results suggest that CH₃ SAM surfaces were the least proinflammatory of all SAM surfaces. Overall, similar trends were observed in the profiles of proteins eluted from surfaces exposed to filtered 10% HS or 1% HP. However, the unique profiles of adsorbed proteins on different SAM surface chemistries may be related to their differential interactions with cells, including immune/inflammatory cells.