Expression of cDNA clones by coupled in vitro transcription/translation and transfection into COS-1 cells: protein mapping in two-dimensional gels.
We present two procedures that can be used to map proteins in two-dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of the in vitro transcription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method--which faithfully reproduces post-translational modifications--is based on the expression of cDNAs transfected into COS-1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha-ras p21 and rab 11, two small GTP-binding proteins known to undergo complex modifications.
Madsen
P
P
Institute of Medical Biochemistry, Aarhus University, Denmark.
Gromov
P
P
Celis
J E
JE
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D005092:Exonucleases; D005095:Exoribonucleases; D050556:Fatty Acid-Binding Proteins; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D011905:Genes, ras / Q000235:genetics*; D018996:Myelin P2 Protein / Q000235:genetics*; D009363:Neoplasm Proteins; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; D014158:Transcription, Genetic; D014162:Transfection; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D005092:Exonucleases; D005095:Exoribonucleases; D050556:Fatty Acid-Binding Proteins; D019204:GTP-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011506:Proteins; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C089242:S100A7 protein, human; C413442:SFN protein, human