The competitive-colchicine-binding assay is based on a scintillation proximity assay technology using bovine brain tubulin, which has been modified so that random surface lysines contain a covalently linked, long-chain biotin derivative (CytoDYNAMIX screen 15, Cytoskeleton Inc.). Briefly, 10 μl of each compound was added to a well of a 96-well plate (Low PB CorningCostar, Amsterdam, The Netherlands). Tritiated colchicine (100 μl; Perkin-Elmer; specific activity 70–80 Ci mmol−1) was added to 300 μl of tubulin-binding buffer and then 10 μl was added in each well. Premix tubulin–biotin–streptavidin scintillation proximity assay beads (180 μl; 4.4 mg streptavidin yttrium silicate beads are mixed into 15 ml buffer and incubated on a slow 10 r.p.m. rotator at 4 °C for 30 min; Amersham Bioscience, Little Chalfont, Buckinghamshire, UK) were added to each well for 3 h at 37 °C. The plates were then read on a scintillation counter (Packard Instrument, Meriden, CT, USA; Topcount Microplate Reader) and the percentage of inhibition was calculated (Tahir et al, 2000 (link)).
Cytodynamix screen15
Manufactured by Cytoskeleton
Sourced in United States
The CytoDYNAMIX Screen15 is a high-throughput screening system designed for the analysis of cellular dynamics. It provides a platform for researchers to study various cellular processes, such as cytoskeletal organization, organelle movement, and cell migration. The system utilizes advanced imaging technologies to capture and analyze live-cell data in a multiwell format, enabling the simultaneous evaluation of multiple experimental conditions.
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7 protocols using cytodynamix screen15
1
Competitive Colchicine-Binding Assay for Tubulin
2
Competitive Colchicine Binding Assay
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Binding of PPP and PPT (both from the Biovitrum compound collection) and colchicine (Sigma C-9754) were performed using colchicine site competitive assay kit Cytodynamix Screen 15 (Cytoskeleton, CDS15), according to the manufacturer's instruction. In separate experiments [3H]-colchicine (American Radiolabeled Chemicals, ART-722), [3H]-PPP (Biovitrum) or [3H]-PPT (Biovitrum) were used as tracers, having specific activities of approximately 3145, 670, and 670 GBq/mmol, respectively. The concentration of [3H]-colchicine in the assay was 53 nM, and the concentration of [3H]-PPP or [3H]-PPT was 4-fold higher, due to lower specific activity, keeping within the limit of the kit procedure as stated by the manufacturer. Compound stock solutions were 10mM in DMSO, and serial dilutions 1:3 were made in DMSO prior to transfer to the assay plate. Top concentration of compound dilution in the assay was 42 μM. Serial dilutions were made in duplicates. Briefly, 10 μL of compound dilution and 10 μL of tracer dilution were incubated with 180 μL of tubulin-biotin SPA-beads for 45 minutes at 37 °C after a short initial shake. Readings were done using a 1450 MicroBeta Trilux 32 BI. Data were analysed using Xlfit 4 parametric linear regression model 205.
3
Colchicine Site Competitive Assay
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Example 6
The affinity of compounds 8c to colchicine binding site was determined using colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., CO, USA) using the standard protocol of the manufacturer to determine Ki values (μM). Biotin-labelled tubulin (0.5 g) in 10 μL of reaction buffer was mixed with [3H]colchicine (0.08 μM, PerkinElmer, Waltham, Mass.) and the test compounds (positive control colchicine, negative control vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96-well plate (final volume: 100 μL). After incubating for 2 h at 37° C. with gentle shaking, streptavidin-labelled yttrium SPA beads (80 g in 20 μL reaction buffer, PerkinElmer, Waltham, Mass.) were added to each well and incubated for 30 min at 4° C. The plates were then read on a scintillation counter (Packard Instrument, Topcount Microplate Reader) and the percentage of inhibition was calculated [54]
4
Benzotriazole Compounds Colchicine Site Assay
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The affinity of Benzotriazole compounds to colchicine binding site was determined using a colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., Denver, CO, USA) using the standard protocol of the manufacturer to determine Ki values (μM). Biotin-labelled tubulin (0.5 μg) in 10 μL of reaction buffer was mixed with [3H]colchicine (0.08 μM, PerkinElmer, Waltham, MA) and the test compounds (positive control colchicine, negative control vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96-well plate (final volume: 100 μL). After incubating for 2 h at 37 °C with gentle shaking, streptavidin-labelled yttrium SPA beads (80 μg in 20 μL reaction buffer, PerkinElmer, Waltham, MA) were added to each well and incubated for 30 min at 4 °C. The plates were then read on a scintillation counter (Packard Instrument, Topcount Microplate Reader) and the percentage of inhibition was calculated [55 (link),56 (link)].
5
Colchicine Site Competitive Assay
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Example 6
The affinity of compounds 8c to colchicine binding site was determined using colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., CO, USA) using the standard protocol of the manufacturer to determine Ki values (μM). Biotin-labelled tubulin (0.5 μg) in 10 μL of reaction buffer was mixed with [3H]colchicine (0.08 μM, PerkinElmer, Waltham, Mass.) and the test compounds (positive control colchicine, negative control vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96-well plate (final volume: 100 μL). After incubating for 2 h at 37° C. with gentle shaking, streptavidin-labelled yttrium SPA beads (80 μg in 20 μL reaction buffer, PerkinElmer, Waltham, Mass.) were added to each well and incubated for 30 min at 4° C. The plates were then read on a scintillation counter (Packard Instrument, Topcount Microplate Reader) and the percentage of inhibition was calculated [54]
6
Colchicine Site Affinity of Quinoline Compounds
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The affinity of selective quinoline compounds 19b , 19h , 20c and 20j as well as CA-4 to colchicine binding site was determined using Colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., Denver, CO, USA) using the standard protocol of the manufacturer to determine Ki values (μM) [47 (link)]. Briefly, selected concentrations of each compound were added to 96-well plate. Tritiated colchicine (100 mL; Perkin-Elmer; specific activity 70–80 Ci mmol−1) was added to 300 mL of tubulin-binding buffer. From this, 10 µL was added in each well. Premix tubulin-biotine-streptavidin scintillation proximity assay beads (180 mL; 4.4 mg streptavidin yttrium silicate beads (Amersham Bioscience, London, UK) are mixed into 15 mL buffer and incubated on a slow 10 r.p.m. rotator at 4 °C for 30 min, and were added to each well for 2 h at 37 °C. The plates were then read on a scintillation counter (Topcount Microplate Reader, Packard Instruments, Winooski, VT, USA) [48 (link),49 (link)].
7
Colchicine Site Binding Assay of Compound 12c
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The affinity of compounds 12c to colchicine binding site was determined using colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., Denver, CO, USA) using the standard protocol of the manufacturer to determine Ki. Biotin-labelled tubulin (0.5 µg) in 10 µL of reaction buffer was mixed with [3H]colchicine (0.08 µM, PerkinElmer, Waltham, MA) and the test compounds (positive control colchicine, negative control vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96-well plate (final volume: 100 µL). After incubating for 2 h at 37 °C with gentle shaking, streptavidin-labelled yttrium SPA beads (80 µg in 20 µL reaction buffer, PerkinElmer, Waltham, MA) were added to each well and incubated for 30 min at 4 °C. The plates were then read on a scintillation counter (Packard Instrument, Topcount Microplate Reader) and the percentage of inhibition was calculated50 (link),51 (link).
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