Inhibition of [3H]colchicine binding to tubulin was measured in 0.1 mL reaction mixtures, each containing 1.0 µM tubulin, 5.0 µM [3H]colchicine (Perkin-Elmer), 5% (v/v) dimethyl sulfoxide, compounds at 1.0 or 5.0 µM, as indicated, and components that stabilize the colchicine binding activity of tubulin (1.0 M monosodium glutamate [adjusted to pH 6.6 with HCl in a 2.0 M stock solution], 0.5 mg/mL bovine serum albumin, 0.1 M glucose-1-phosphate, 1.0 mM MgCl2, and 1.0 mM GTP). Incubation was for 10 min at 37 °C, when in control reaction mixtures colchicine binding is 40–60% complete. Reactions were stopped with 2.0 mL of ice-cold water, with the reaction mixtures being placed on ice. Each diluted sample was poured onto a stack of two DEAE-cellulose filters (GE Biomedical), followed by 3 successive 2 mL aliquots of ice-cold water. A reduced vacuum was used to remove excess water from the filters, which were washed three times with 2 mL water and placed into vials containing 5 mL of Biosafe II scintillation cocktail. Samples were counted 18 h later in a Beckman scintillation counter. Samples with inhibitors were compared to samples with no inhibitor, and percent inhibition was determined, correcting all values for the amount of radiolabel bound to the filters in the absence of tubulin.
3h colchicine
Manufactured by PerkinElmer
Sourced in United States
[3H]colchicine is a radiolabeled compound used in scientific research. It is a derivative of the natural compound colchicine, with a tritium (3H) label incorporated into its structure. This product is commonly utilized in various experimental applications, such as binding studies and cell biology research, where the radioactive signal can provide valuable insights.
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Lab products found in correlation
Colchicine, by PerkinElmer (6 mentions)
Cytodynamix screen15, by Cytoskeleton (4 mentions)
Yttrium spa beads, by PerkinElmer (4 mentions)
Colchicine, by Cytoskeleton (2 mentions)
Scintillation counter, by Beckman Coulter (1 mentions)
Paclitaxel taxol, by Merck Group (1 mentions)
Colchicine, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Mdr 1, by Santa Cruz Biotechnology (1 mentions)
Vinblastine, by Merck Group (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
7 protocols using 3h colchicine
1
Colchicine Binding Inhibition Assay
2
Colchicine Binding Site Assay of G-1 in Tubulin
3
Chemotherapeutic agents and cytoskeleton
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Nocodazole, paclitaxel (Taxol), vinblastine, and colchicine were purchased from Sigma-Aldrich (St. Louis, MO). SRF was purchased from ChemDiv while SB203580, PD98059 and SP600125 were from Calbiochem (San Diego, CA). Antibodies were obtained from the following companies: vimentin, α- and β-tubulin, JNK-1, Mdr-1, Bcl-2, pBcl-2 (T56), pBcl-2 (S70), pBcl-2 (S87) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); pp38, pERK1/2, pJNK (Cell Signaling Technology, Denver, MA); monoclonal anti P-glycoprotein (MDR) (Sigma, USA) and monoclonal actin antibodies were from BD Pharmingen (San Diego, CA). [3H]vinblastine (specific activity, 11.6 Ci/mmol) and streptavidin-coated yttrium silicate scintillation proximity assay (SPA) beads were purchased from GE Healthcare (Buckinghamshire, UK). [3H]colchicine (specific activity, 80.4 Ci/mmol) was obtained from PerkinElmer (Boston, MA, USA).
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
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)].
6
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]
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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