Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin

Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain....

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Veröffentlicht in:BioTechniques. - 1993. - 29(2000), 1 vom: 17. Juli, Seite 156-60
1. Verfasser: Tahir, S K (VerfasserIn)
Weitere Verfasser: Kovar, P, Rosenberg, S H, Ng, S C
Format: Aufsatz
Sprache:English
Veröffentlicht: 2000
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Aminophenols Bibenzyls Solvents Stilbenes Sulfonamides Tubulin Tritium 10028-17-8 E 7010 mehr... 141430-65-1 Yttrium 58784XQC3Y combretastatin 7O62J06F18 Mebendazole 81G6I5V05I Streptavidin 9013-20-1 Podophyllotoxin L36H50F353 Colchicine SML2Y3J35T Demecolcine Z01IVE25KI
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500 |a Date Revised 28.09.2018 
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520 |a We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used 
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700 1 |a Rosenberg, S H  |e verfasserin  |4 aut 
700 1 |a Ng, S C  |e verfasserin  |4 aut 
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