首页>
外国专利>
TRIVALENT METAL MEDIATED HOMOGENEOUS LUMINESCENT PROXIMITY ASSAY
TRIVALENT METAL MEDIATED HOMOGENEOUS LUMINESCENT PROXIMITY ASSAY
展开▼
机译:三价金属介导的均质发光邻近测定
展开▼
页面导航
摘要
著录项
相似文献
摘要
An in vitro protein kinase assay technology that (1) exhibits a high assay signal to background ratio (S/B) and range (S-B); (2) is homogenous; (3) is non-radioactive; and (4) does not require a phospho-specific antibody. Said assay involves complexing a trivalent metal ion (e.g. Ga3+, Fe3+, Al3+, In3+, Ru3+, Sc3+, Y3+) to the surface of amplified luminescent proximity assay acceptor or donor beads, e.g., via a suitable linker such as nitrilotriacetic acid (NTA; also referred to as carboxymethyl-lysine), iminodiacetic acid (IDA), or an appropriately substituted N-containing heterocycle, for example a triazoheterocycle, for example a triazocyclononaneononane, such as 1-propylamino-4-acetato-1,4,7-triazacyclononane. A protein (or constituent part) or other kinase substrate is bound to the surface of the other of an amplified luminescent proximity assay acceptor or donor bead and, if phosphorylated, brought into proximity with the trivalent metal ion-complexed acceptor bead to generate a luminescent signal. Presence of a kinase inhibitor inhibits phosphorylation and therefore signal generation and, in this way, is detectable. As the invention described herein recognizes the presence or absence of phosphate groups on a protein, (or constituent part), or other biological macromolecule (e.g., mono, di, or trinucleotides, cyclic nucleotides or phosphate substituted inositols), it is broadly applicable to any phosphorlylation or dephosphorylation reaction enzymes and provides a highly robust and flexible assay format for protein kinases and other enzyme classes, including lipid kinases, phosphatases, phosphodiesterases and others.
展开▼