Multicolor in vitro translation.
In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL-labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL-lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro-translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.
Traverso
Giovanni
G
Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center, and Graduate Program in Human Genetics, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, Maryland 21231, USA.
Diehl
Frank
F
Hurst
Robin
R
Shuber
Anthony
A
Whitney
Duncan
D
Johnson
Constance
C
Levin
Bernard
B
Kinzler
Kenneth W
KW
Vogelstein
Bert
B
D001896:Boron Compounds; D015179:Colorectal Neoplasms / Q000235:genetics*; D020552:Comet Assay / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D005243:Feces / Q000737:chemistry*; D006801:Humans; D014176:Protein Biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004273:DNA, Neoplasm