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In addition to the fully activated, diphosphorylated ERK1\/2 protein, monophosphorylated forms of ERK1\/2 have been observed, which may have distinct biological functions. We report here on the highly sensitive detection and differentiation of unphosphorylated, threonine-phosphorylated (pT), tyrosine-phosphorylated (pY) and diphosphorylated ERK1 and ERK2 by capillary isoelectric focusing followed by immunological detection (CIEF-immunoassay). Eight different phosphorylated and unphosphorylated forms of ERK1\/2 were resolved according to charge. The unequivocal identification and differentiation of ERK1 and ERK2 forms monophosphorylated at either threonine or tyrosine was achieved by competitive blocking with specific phospho-peptides and different phosphorylation-sensitive antibodies. The suitability of the additional pT-ERK1\/2 and pY-ERK1\/2 differentiation for the time-resolved in-depth study of phospho-form distribution in response to specific stimuli is demonstrated in human neuroblastoma SH-SY5Y and monocytic THP-1 cell lines and in human peripheral blood mononuclear cells.<\/jats:p>","DOI":"10.1038\/srep12767","type":"journal-article","created":{"date-parts":[[2015,8,3]],"date-time":"2015-08-03T10:28:39Z","timestamp":1438597719000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Detection and Differentiation of Threonine- and Tyrosine-Monophosphorylated Forms of ERK1\/2 by Capillary Isoelectric Focusing-Immunoassay"],"prefix":"10.1038","volume":"5","author":[{"given":"Inga","family":"Kraus","sequence":"first","affiliation":[]},{"given":"Daniela","family":"Besong Agbo","sequence":"additional","affiliation":[]},{"given":"Markus","family":"Otto","sequence":"additional","affiliation":[]},{"given":"Jens","family":"Wiltfang","sequence":"additional","affiliation":[]},{"given":"Hans","family":"Klafki","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2015,8,3]]},"reference":[{"key":"BFsrep12767_CR1","doi-asserted-by":"publisher","first-page":"PE45","DOI":"10.1126\/stke.2512004pe45","volume":"2004","author":"EC Cheung","year":"2004","unstructured":"Cheung, E. 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