Two-dimensional electrophoresis of protein from cultured postimplantation rat embryos for developmental toxicity studies.
A simple method for two-dimensional electrophoresis (2-DE) of rat embryonic protein was described. Rat embryos cultured for 24h from day 10.5 of gestation were used as protein samples. Protein samples were lysed in rehydration buffer and separated by isoelectric focusing with immobilized pH gradient for the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the second dimension. The use of the DeStreak Reagent as an antioxidant in the lysis buffer and electrode pads in the isoelectric focusing greatly improved the 2-DE pattern. When an embryo was used as a protein sample, about 800 protein spots were detected by silver staining in a 2-DE gel of the standard format. Eighty-one protein spots were identified by mass spectrometry for a primary 2-DE map. The same method could be applied to yolk sac membranes from the cultured embryos. The present method was considered to be suitable for a concomitant 2-DE analysis in in vitro developmental toxicity studies.
Usami
Makoto
M
Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan. usami@nihs.go.jp
Mitsunaga
Katsuyoshi
K
Nakazawa
Ken
K
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004622:Embryo, Mammalian / Q000737:chemistry / Q000196:embryology / Q000378:metabolism*; D047108:Embryonic Development / Q000502:physiology*; D009924:Organ Culture Techniques; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D015017:Yolk Sac / Q000737:chemistry / Q000196:embryology / Q000378:metabolism
D011506:Proteins