Radioactive labeling of proteins in cultured postimplantation mouse embryos. I. Influence of the embryo preparation method.
Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode's solution containing [3H]amino acids (mixture) at a concentration of 27 microCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo "naked"); d) naked embryos cut randomly into pieces (Day 10 embryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked embryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first method (see above) fulfilled the conditions required at the best.
Nowak
J
J
Institut für Humangenetik, Freie Universität Berlin, FRG.
Klose
J
J
D000596:Amino Acids / Q000378:metabolism*; D000650:Amnion / Q000502:physiology; D000818:Animals; D046508:Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development; D005260:Female; D005865:Gestational Age; D051379:Mice; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D014176:Protein Biosynthesis; D013048:Specimen Handling; D014316:Tritium; D015017:Yolk Sac / Q000502:physiology
D000596:Amino Acids; D014316:Tritium