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A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single‐pulse UV Laser Irradiation
Author(s) -
Mutskov Vesco,
Angelov Dimitar,
Pashev Lliya
Publication year - 1997
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1997.tb03136.x
Subject(s) - dna , cuvette , chromatin , irradiation , histone , covalent bond , chemistry , laser , macromolecule , biophysics , pulse (music) , biochemistry , biology , optics , organic chemistry , physics , detector , nuclear physics
— 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.