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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Ares, Manuel |
| Abstract | In vitro splicing studies are a powerful means of investigating the requirements and mechanisms of action of the many components of the splicing apparatus. The ability to add and subtract components, purify activities, and reconstitute activity, as well as to expose the apparatus to chemical probes of various types, allows a far more mechanistically detailed view of the process to emerge than is available from genetic or in vivo studies alone. Two kinds of activities are assayed during in vitro splicing. The first concerns the chemical conversion of the substrate pre-mRNA into splicing intermediates and products and is usually visualized using a labeled substrate followed by separation on a denaturing gel. The second concerns the assembly of noncovalent complexes between the substrate and the myriad components of the splicing apparatus. This is also visualized using a labeled substrate, but the separation of complexes is achieved using native gel electrophoresis or gradient sedimentation. In this protocol, we describe the splicing reaction and its preparation for analysis by denaturing gels and native splicing complex gels. We also provide conditions for depletion of ATP, a critical cofactor that is hydrolyzed during numerous key steps in spliceosome assembly and splicing progression. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 10 |
| Volume Number | 2013 |
| e-ISSN | 15596095 |
| Journal | Cold Spring Harbor Protocols |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2013-10-01 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Biochemistry Methods Complex Mixtures Isolation & Purification Metabolism Rna Splicing Saccharomyces Cerevisiae Enzymology Adenosine Triphosphate Analysis Electrophoresis, Polyacrylamide Gel Molecular Biology Journal Article |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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