Detection of four new single-stranded telomeric DNA binding proteins by means of an optimized protein blotting procedure

Abstract

The preservation and replication of telomeres are likely to involve multiple protein interactions. We describe a procedure for detecting sequence-specific telomeric DNA binding proteins in crude nuclear extracts. The technique involves electrophoretic transfer of SDS-PAGE fractionated crude nuclear proteins onto PVDF membranes with subsequent incubation in 2% (wt/vol) bovine serum albumin blocking solution. Incubation of the blocked filters with a 5'-biotin-labeled telomeric DNA probe under optimal binding conditions and subsequent biotin detection by means of peroxidase-linked streptavidin complexes reveals sequence-specific protein-telomeric DNA interactions. Using this technique, we identified 13 proteins that specifically bind the single-stranded telomere repeats of (TTAGGG)n, four of which have not been characterized as telomere binding so far.