Ultrasensitive fluorescence-based detection of nascent proteins in gels.
The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine. We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels. A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification. Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator. Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner. The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing. The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening.
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AmberGen, Inc., 1106, Commonwealth Avenue, Boston, Massachusetts, 02215, USA.
Mamaev
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Olejnik
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Rothschild
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D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D001896:Boron Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D012358:RNA, Transfer, Met / Q000378:metabolism; D012680:Sensitivity and Specificity
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001426:Bacterial Proteins; D001896:Boron Compounds; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D011506:Proteins; D012358:RNA, Transfer, Met