Chloramphenicol acetyltransferase assay.
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.
Smale
Stephen T
ST
D000105:Acetyl Coenzyme A / Q000378:metabolism; D019677:Artificial Gene Fusion; D001345:Autoradiography / Q000379:methods*; D002250:Carbon Radioisotopes / Q000378:metabolism; D002457:Cell Extracts / Q000302:isolation & purification; D002701:Chloramphenicol / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000235:genetics; D002855:Chromatography, Thin Layer; D003584:Cytological Techniques / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013194:Staining and Labeling / Q000379:methods*; D014158:Transcription, Genetic; D014162:Transfection
D002250:Carbon Radioisotopes; D002457:Cell Extracts; D029968:Escherichia coli Proteins; D002701:Chloramphenicol; D000105:Acetyl Coenzyme A; D015500:Chloramphenicol O-Acetyltransferase