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Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte
Author(s) -
Tate Suresh S.,
Urade Reiko,
Micanovic Radmila,
Gerber Louise,
Udenfriend Sidney
Publication year - 1990
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.4.2.2298343
Subject(s) - xenopus , oocyte , alkaline phosphatase , placental alkaline phosphatase , messenger rna , cloning (programming) , biology , microbiology and biotechnology , translation (biology) , phosphatase , gene expression , chemistry , biochemistry , gene , enzyme , embryo , phosphorylation , computer science , programming language
The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.—Tate, S. S.; Urade, R.; Micanovic, R.; Gerber, L.; Udenfriend, S. Secreted alkaline phosphatase: an internal marker for expression of injected mRNAs in the Xenopus oocyte. FASEB J. 4: 227‐231; 1990.