Detection of four new single-stranded telomeric DNA binding proteins by means of an optimized protein blotting procedure.
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.
Kellner
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U
Center of Pathology, University of Kiel, Germany. udo.kellner@medizin.uni-magdeburg.de
Haenssle
H A
HA
Rudolph
P
P
Parwaresch
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R
D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D004277:DNA, Single-Stranded / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D020080:Tandem Repeat Sequences; D016615:Telomere / Q000235:genetics*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins