| Chapter title |
Purification of RNA–Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture
|
|---|---|
| Chapter number | 5 |
| Book title |
RNA-Protein Complexes and Interactions
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3591-8_5 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3589-5, 978-1-4939-3591-8
|
| Authors |
Naoyuki Kataoka, Kataoka, Naoyuki |
| Abstract |
In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 7 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 3 | 43% |
| Professor > Associate Professor | 1 | 14% |
| Researcher | 1 | 14% |
| Student > Master | 1 | 14% |
| Unknown | 1 | 14% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 3 | 43% |
| Agricultural and Biological Sciences | 1 | 14% |
| Neuroscience | 1 | 14% |
| Medicine and Dentistry | 1 | 14% |
| Unknown | 1 | 14% |