Phosphotyrosine Content in Mitogen-Activated Protein Kinase

Measurement of Phosphotyrosine Content in Mitogen-Activated Protein Kinase 

MAPK is active only when phosphorylated on two specific amino acids, one threonine and one tyrosine. Because of this, MAPK activity can be measured by determining the content of phosphotyrosine in the enzyme using highly specific antiphosphotyrosine antibodies. Known amounts of protein are separated by polyacrylamide gel electrophoresis, transferred to sheets of nitrocellulose (or polyvinylidine difluoride), and probed with antiphosphotyrosine antibodies. To increase the signal produced by the detection system, it may be necessary to immunoprecipitate MAPK from known quantities of tissue, or to partially purify MAPK, biochemically, before immunoblot analysis. An increase in specific antiphosphotyrosine antibody binding implies that MAPK activity increases. 

This technique has the advantage that it is relatively simple to perform. However, comparisons among successive experiments are dependent on several factors. These factors include the efficiency of protein transfer 

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