The ability of G-1 binding to the colchicine binding site in tubulin was examined using a CytoDYNAMIX screen 15 assay kit (Cytoskeleton Inc., Denver, CO, USA) in accordance with manufacturer’s instruction and previous description. Biotin-labeled 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-labeled 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 radioactivity was determined using Packard TopCount Microplate Scintillation Counter (Packard Instrument, Meriden, CT, USA).
Colchicine
Manufactured by PerkinElmer
Sourced in France, United States
Colchicine is a laboratory compound used in scientific research. It is a naturally occurring alkaloid found in certain plants, such as the autumn crocus. Colchicine is commonly used as a tool in cell biology and genetics research to study processes like cell division and chromosome structure.
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Lab products found in correlation
3h colchicine, by PerkinElmer (6 mentions)
Cytodynamix screen15, by Cytoskeleton (4 mentions)
Yttrium spa beads, by PerkinElmer (4 mentions)
Colchicine, by Cytoskeleton (2 mentions)
Liquid scintillation counter, by Beckman Coulter (1 mentions)
Ultima gold scintillant, by PerkinElmer (1 mentions)
Scintillation counter, by Beckman Coulter (1 mentions)
4 ml quartz cuvette, by Hellma (1 mentions)
Colchicine, by AppliChem (1 mentions)
Ls50b, by PerkinElmer (1 mentions)
Originpro, by OriginLab (1 mentions)
8 protocols using colchicine
1
Colchicine Binding Site Assay of G-1 in Tubulin
2
Tubulin-Colchicine Binding Assay
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The tubulin was prepared from bovine brain as previously described [77 (link)]. Pure tubulin (3 µM final concentration) in cold PIPES-EGTA (PME) buffer (100 millimolar (mM) piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 1 mM magnesium chloride (MgCl2), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 6.65) was mixed at 4 °C with a mix of [3H]-colchicine (82.6 Ci/mmol) (Perkin-Elmer, Courtaboeuf, France, 50 nM final concentration) and the competitor ESE-16 (100 µM final concentration) in a final volume of 200 µL. Following a 30-min incubation at 30 °C, the samples were deposited onto 50 µL of pre-sedimented DEAE Sephadex A25 in BRB80 buffer. All subsequent steps were carried out at 4 °C. Samples were incubated for 10 min with continuous agitation to ensure quantitative binding of tubulin to the gel. Following centrifugation (2400× g, 4 min), supernatants were discarded and the pellets containing the bound molecule-tubulin complexes were washed four times with 1 mL of PME buffer. Pellets were incubated for 10 min with 500 µL of ethanol to solubilize the tubulin-bound tritiated colchicine and 400 µL aliquots of the ethanol solutions were transferred to 5 mL of Ultima Gold scintillant (Perkin-Elmer, Courtaboeuf, France) for determination of radioactivity using a liquid scintillation counter (Beckman Coulter, Brea, CA, USA).
3
Colchicine Binding Inhibition Assay
4
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]
5
Calcium-Dependent Colchicine Binding Affinity
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To assess the influence of Ca2+ on the affinity of D6.2 towards colchicine, the equilibrium dissociation constant (Kd) was measured by fluorescence titration using an LS50B spectrophotometer (Perkin Elmer, Waltham, Massachusetts, USA) as described previously (Barkovskiy et al., 2019 ▸ ). In brief, 2 ml purified D6.2-His6 at 100 nM in 10 mM HEPES–NaOH pH 7.5, 50 mM NaCl with or without the addition of 200 mM CaCl2 was equilibrated in a 4 ml quartz cuvette (Hellma Analytics, Müllheim, Germany) for 10 min at ambient temperature. A stock solution of 40 µM colchicine (Applichem, Darmstadt, Germany) in the same buffer was then added stepwise in 0.25–4 µl aliquots, followed by stirring for 60 s. The tyrosine and tryptophan fluorescence (excitation at 280 nm, emission at 345 nm) was measured at each colchicine concentration for 15 s under stirring. The inner filter effect, due to the absorption of colchicine at 345 nm, was corrected by performing a control titration of N-acetyltryptophanamide as described by Vopel et al. (2005 ▸ ). The resulting data were fitted according to bimolecular complex formation (Barkovskiy et al., 2019 ▸ ) with Origin Pro (OriginLab, Northampton, Massachusetts, USA).
6
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)].
7
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]
8
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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