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Published on: February 22, 2014
Affinity Purification of Embryo Proteins Phosphorylated on Tyrosine In Vitro*: (phosphotyrosine/protein
Wanping Jiang1, Gerard Peaucellier1, William H Kinsey1
1Department of Anatomy and Cell Biology, University of Miami School of Medicine, Miami, Florida 33101.
Abstract:
Fertilization of the sea urchin egg is accompanied by activation of one or more protein tyrosine kinases which have been shown to phosphorylate a restricted set of egg proteins in vitro. In order to characterize these tyrosine kinase substrates, we have used an antibody specific for phosphotyrosine to prepare an immunoaffinity column capable of binding phosphotyrosine containing proteins. This column bound five 32 P-labelled proteins from detergent extracts of embryo membranes phosphorylated in vitro. All were very tightly membrane associated, requiring detergent solubilization. Phosphoamino acid analysis revealed that each of these proteins was phosphorylated exclusively on tyrosine, indicating that they do not act as substrates for other classes of protein kinases.
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