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A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single-pulse UV Laser Irradiation
Ist Teil von
Photochemistry and photobiology, 1997-07, Vol.66 (1), p.42-45
Ort / Verlag
Oxford, UK: Blackwell Publishing Ltd
Erscheinungsjahr
1997
Link zum Volltext
Quelle
Wiley Online Library All Journals
Beschreibungen/Notizen
— Photocrosslinking of proteins to DNA by single‐pulse UV laser has been used only in analytical experiments, carried out with reconstituted complexes of a single DNA binding protein and a labeled target sequence. Here we propose a large‐scale technique for irradiation of nuclei, generating preparative quantities of covalently linked protein‐DNA complexes for further analysis of the partner molecules. The use of a flow cuvette allows a milligram of DNA in either nuclei or chromatin to be irradiated by a single pulse within few minutes. The efficiency of crosslinking varies from 6 to 12% of the total nuclear proteins. The presence of histones and other chromosomal proteins in the crosslinked protein‐DNA complexes was demonstrated by using specific antibodies. The irradiation procedure can be fully automated using a microcomputer.