Efficient electroblotting of gel-resolved proteins onto diamond-like carbon-coated plate for protein-chip

Abstract

We have developed a technique, which can produce high-density protein chips. In this technique, proteins are separated by gel electrophoresis, and electroblotted by semidry blotting apparatus onto a diamond-like carbon-coated stainless-steel plate (DLC plate) of which surface is modified chemically with amino groups. Proteins immobilized on this protein chip can interact with other proteins in solution, and proteins interacted with the immobilized proteins can be identified by mass spectrometric analysis. However, the electroblotting efficiency was not stable. We anticipated that the unstable efficiency might be related to the fluctuation of temperature during electroblotting. In the present study, to investigate the optimal temperature for efficient and effective electroblotting, we developed a semidry blotting apparatus, which could regulate blotting temperature from 4 to 45°C (±1°C). The high and reproducible blotting efficiency was obtained at not low temperature but rather high temperature (30°C). The temperature did not impede protein identification or analysis of protein-protein interactions on the DLC plates by mass spectrometry.