Fluorescent microplate-based analysis of protein-DNA interactions II:immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Roman Catholicism, diplomacy, and the European communities, 1958-1964

This article investigates the Roman Catholic Church's role in the process of European integration from the first Hallstein Commission in 1958 to the failure of the Holy See's application to establish a diplomatic representation at the European Economic Community in 1964. The article focuses on the Church's response toward emerging European institutions and shows that local mobilization in Luxembourg, Strasbourg, and Brussels was instrumental in shaping relations between the Catholic Church and the European Communities (EC). The Church's position toward the EC, placing local communities as prime actors in dialogue with European institutions, reflected the sensitive nature of religion during the Cold War.