UV crosslinking of proteins to nucleic acids.
Irradiation of protein-nucleic acid complexes with ultraviolet light causes covalent bonds to form between the nucleic acid and proteins that are in close contact with the nucleic acid. Thus, UV crosslinking may be used to selectively label DNA-binding proteins based on their specific interaction with a DNA recognition site. As a consequence of label transfer, the molecular weight of a DNA-binding protein in a crude mixture can be rapidly and reliably determined. This unit provides 3 protocols for executing DNA-protein crosslinking; one uses the halogenated thymidine analog bromodeoxyuridine (BrdU) to produce a DNA probe that is especially sensitive to UV-induced crosslinking. An alternate protocol describes crosslinking using a non-BrdU substituted probe, and another alternate protocol provides a method for in situ crosslinking.
Chodosh
L A
LA
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001973:Bromodeoxyuridine; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D003851:Deoxyribonucleases; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008970:Molecular Weight; D010777:Photochemistry / Q000379:methods*; D011485:Protein Binding; D014466:Ultraviolet Rays
D015342:DNA Probes; D004268:DNA-Binding Proteins; D004247:DNA; D003851:Deoxyribonucleases; D001973:Bromodeoxyuridine