DIGOXIGENIN-LABELED PEPTIDES FOR THE IMMUNOLOGICAL QUANTIFICATION OF INTRACELLULAR SIGNALING PROTEINS - APPLICATION TO THE MAP KINASE KINASE ISOFORM MEK2

Two competitive enzyme immunoassays using digoxigenin-labeled peptides have been devloped for the quantification of the protein kinase MEK2 in cell extracts. Rabit polyclonal antibodies directed against wither the amino-terminal or proline-rich amino acid sequences of MEK2 were u sed for the immunoconcentration of the protein. Anti-digoxigenin Fab f ragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoass ays exhibited a sensitivity level of 63 and 71 fmol/mL, respectively, and displayed a half-maximal saturation value of 1320 and 1780 fmol/mL . The intra- and inter-assay coefficients of variation for both assays assessed at three different concentrations of MEK2 were lower than 6% and 12%, respectively. The amount of MEK2 measured by the two methods demonstrated an excellent correlation with the expression level of th e protein detected by immunoblot analyses when rested on different cel l lysates.