A preparative method for crosslinking proteins to DNA in nuclei by single-pulse UV laser irradiation.
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.
Mutskov
V
V
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Angelov
D
D
Pashev
I
I
D000818:Animals; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm / Q000378:metabolism* / Q000528:radiation effects; D005622:Friend murine leukemia virus; D006657:Histones / Q000378:metabolism* / Q000528:radiation effects; D007834:Lasers; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D011485:Protein Binding; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D006657:Histones