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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Rio, Donald C. |
| Abstract | One of the oldest and simplest (and still very useful) methods for detecting RNA-protein interactions is the filter-binding assay. If a mixture of RNA and protein is passed through a nitrocellulose filter, the protein will be retained and the RNA will pass through. But if the protein is capable of binding RNA, then RNA will be retained on the filter as well. This protocol requires a purified protein (or chromatographic fractions) of interest and (32)P-labeled RNA. To perform the assay, the protein sample is serially diluted to several concentrations. It is then mixed with a fixed amount of radioactive RNA and allowed to bind under desired conditions for 30-60 min. The binding reactions are then applied to a 96-well dot-blot apparatus with low vacuum to trap the complexes on three membranes: The top membrane traps aggregates, the middle membrane (nitrocellulose) binds proteins and RNA-protein complexes, and the bottom membrane (which is charged) collects free RNA. After washing and drying, the membranes are exposed to phosphor-imaging screens for quantitation. Alternatively, single, larger filters (that can be counted in a scintillation counter) and a filter manifold can be used. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 10 |
| Volume Number | 2012 |
| e-ISSN | 15596095 |
| Journal | Cold Spring Harbor Protocols |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2012-10-01 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Rna-binding Proteins Metabolism Rna Adsorption Collodion Chemistry Isotope Labeling Micropore Filters Phosphorus Radioisotopes Protein Binding Journal Article |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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