Detection of ERK activation by a novel monoclonal antibody.
The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.
Yung
Y
Y
Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, Israel.
Dolginov
Y
Y
Yao
Z
Z
Rubinfeld
H
H
Michael
D
D
Hanoch
T
T
Roubini
E
E
Lando
Z
Z
Zharhary
D
D
Seger
R
R
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D004331:Drosophila melanogaster; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D056890:Eukaryota; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015398:Signal Transduction; D018411:Spodoptera; D013912:Threonine; D014162:Transfection; D014443:Tyrosine
D000911:Antibodies, Monoclonal; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases