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Tubulin ligand competition assay (CDS15-P)

Tubulin ligand competition assay (CDS15-P)

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1000 assays (Cat. # CDS15-P) +$1,750.00

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Product Uses Include

Screening compounds for competition to known ligand sites on the tubulin monomer protein.
Screening colchicine binding site analogs
Screening vinblastine binding site analogs
Screening paclitaxel binding site analogs
Screening for cancer cell tubulin selective ligands

Introduction
CytoDYNAMIX Screen™ 15 is designed to detect and measure a compound’s affinity for a drug binding binding site of tubulin such as the colchicine, vinblasine or paclitaxel binding site. CDS15P can be used as a primary or secondary screen. It is economical when compared to the standard microtubule polymerization assay but it is limited by being restricting to one drug binding site.

The CDS15P assay is based on a publication by Tahir and coworkers (1) and takes advantage of Scintillation Proximity Assay (SPA) technology provided by GE Healthcare, Inc. As such, this kit contains the non-proprietary items such as biotin tubulin, buffers, positive and negative controls and protocols. Items not included in the kit are plates, SPA beads and tritiated radio-ligand. These extra items are listed under the Equipment Needed section.

SPA technology requires a close association between a solid phase scintillant (the beads) and the radio-ligand for a signal to be emitted and subsequently detected. As shown in Figure 1, biotin tubulin is the reagent that brings the radio-ligand and the scintillant into close association. If the radio-ligand is tritiated e.g. colchicine then the amplitude of signal is proportional to the number of colchicine binding sites that are occupied by this radio ligand. When you add a competitor of radio-labeled colchicines, for example cold (unlabeled) colchicine, to the mixture then the amplitude of signal will be decreased proportionately with concentration of competitor. In some cases the inhibition can be transitory, for example with nocodazole, the reason being that colchicine binds more tightly than nocodazole but also more slowly than nocodazole, so over a long time frame (>45min) nocodazole is effectively replaced by colchicine.

Figure 1: Priciple of the Tubulin ligand binding assay. The two wells illustrate the situations of (1) no binding competition for the colchicine binding site on tubulin, which yields a high signal and (2) partial competition for the colchicine binding site, in which a lower signal is detected.

Kit contents
The kit contains sufficient reagents for 1000 assays. Please inquire for significant discounts on larger orders. The following components are included:

Biotin tubulin (Cat. # T333P)
General tubulin buffer (Cat. # BST01).
GTP solution (Cat. # BST06).
Control compound for competition.
Detailed instructions and trouble-shooting manual.

Equipment needed

Tritiated tubulin binding compound (e.g. American Radio-label Chemical Co. or GE Healthcare))
Streptavidin SPA beads (GE Healthcare)
96-well plates, low protein binding
Scintillation counter for 96-well plates

Example results
A competition assay for the colchicine binding site (using tritiated colchicine) was performed with ranging concentrations of cold colchicine and paclitaxel (taxol). Cold colchicine had an IC50 of 1.26 µM while paclitaxel, as expected, showed no binding competition in the range tested.

Figure 2: Colchicine competition assay. Cold colchicine and paclitaxel in concentrations between 0.01 and 100 µM were allowed to compete with tritiated colchicine in binding to tubulin. Compunds were incubated with biotin tubulin and streptavidin SPA beads and each well was read for 10 s.

References

Tahir, S. K., Kovar, P., Rosenberg, S. H. and Ng, S. C. (2000). Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin. Biotechniques 29, 156-160

Tubulin ligand competition assay (CDS15-P)

Tubulin ligand competition assay (CDS15-P)

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