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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Smale, Stephen T. |
| Abstract | When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. In this protocol, cells transfected with an Escherichia coli transposon chloramphenicol acetyltransferase (CAT) reporter plasmid are lysed by repeated cycles of freezing and thawing and cellular debris is removed by centrifugation. The lysate is incubated with [(14)C]chloramphenicol and acetyl-coenzyme A; CAT catalyzes the acetylation of chloramphenicol. The acetylated products and the unmodified reactants are separated from the aqueous solution by organic extraction with ethyl acetate. Acetylation is monitored by autoradiography following thin-layer chromatography (TLC) to separate the acetylated from the unacetylated forms. The percent conversion of [(14)C]chloramphenicol to acetyl-[(14)C]chloramphenicol can be measured by PhosphorImager analysis of the TLC plate, by excising the radioactive spots from the TLC plate and counting in a scintillation counter, or by densitometry analysis of an autoradiograph. The acetylated (14)C-labeled product can also be quantified without TLC by organic extraction and scintillation counting using reagent-grade chemicals. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 5 |
| Volume Number | 2010 |
| e-ISSN | 15596095 |
| Journal | Cold Spring Harbor Protocols |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2010-05-01 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Autoradiography Methods Chloramphenicol O-acetyltransferase Analysis Cytological Techniques Gene Expression Genes, Reporter Staining And Labeling Acetyl Coenzyme A Metabolism Artificial Gene Fusion Carbon Radioisotopes Cell Extracts Isolation & Purification Chloramphenicol Genetics Chromatography, Thin Layer Escherichia Coli Proteins Plasmids Promoter Regions, Genetic Protein Biosynthesis Transcription, Genetic Transfection Journal Article |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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