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Identification of RNA Binding Proteins by UNIT 27.2
UV Cross-Linking
UV cross-linking is a standard method used to detect RNA-binding proteins. This method
takes advantage of the ability of a photoreactive group, upon UV irradiation, to trigger
the formation of a covalent bond between the RNA and closely interacting proteins. In
this assay, proteins and a 32P-radiolabeled RNA substrate are incubated to allow complex
formation. The reaction mixture is then UV-irradiated, followed by treatment with RNase.
This produces a protein attached to a short oligoribonucleotide. These protein-oligori-
bonucleotide products are analyzed by standard SDS(UNIT 10.2A) or two-dimen-
sional gel electrophoresis (UNITS 10.3 10.4), followed by phosphorimaging (APPENDIX 3A).
Combined with immunoblotting (UNIT 10.8), immunoprecipitation (UNIT 10.16), and/or mass
spectrometry (UNITS 10.21 10.22), the proteins can often be identified. Site-specific labeling
of the RNA with 32P increases the power of the UV-cross-linking assay because the
RNA-protein interaction site can be defined on the RNA.
The following protocol demonstrates this method using the spliceosomal complex as an
example.
CAUTION: Radioactive materials require special handling. See APPENDIX 1F and the
institutional Radiation Safety Office for guidelines concerning proper handling and
disposal.
NOTE: Extreme caution should be taken to avoid RNase contamination. The experimenter
should always wear gloves and all the tubes and tips that come into contact with the sample
should be certified RNase free; however, DEPC treatment (UNIT 4.1) of solutions and
apparatuses is not necessary.
UV CROSS-LINKING USING A UNIFORMLY LABELED BASIC
RADIOACTIVE RNA PROT
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