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 developed for the quantification of the protein kinase MEK2 in cell extracts. Rabbit polyclonal antibodies directed against either the amino-terminal or proline-rich amino acid sequences of MEK2 were used for the immunoconcentration of the protein. Anti-digoxigenin Fab fragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoassays 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 the protein detected by immunoblot analyses when tested on different cell lysates.
Blais
C
C
Université de Montréal, Quebec, Canada.
Drapeau
G
G
Meloche
S
S
Morais
R
R
Adam
A
A
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D001667:Binding, Competitive / Q000276:immunology; D002460:Cell Line; D002645:Chickens; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism*; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D007424:Intracellular Fluid / Q000201:enzymology*; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D011392:Proline / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000276:immunology; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000276:immunology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010455:Peptides; D011506:Proteins; D011392:Proline; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D004076:Digoxigenin