In vitro tubulin polymerization assay was performed using the fluorescence-based tubulin polymerization kit purchased from Cytoskeleton, Inc. (Denver, CO). The polymerization assay is a one-step assay, which uses a half area 96-well plate (Corning Costar, Corning, NY) for measuring tubulin polymerization. A 5 μL aliquot of a 10× concentration of the test compound in DI was pipetted into each well of the pre-warmed 96-well plates at 37 °C. Following the addition of compound, 50 μL of tubulin MIX was added (as per manufacturer’s protocol). Then, the plate was immediately subjected to kinetic measurements on a SpectraMAx M5e fluorescence microplate reader (Molecular Device, San Jose, CA). The kinetics of tubulin polymerization reaction was monitored for every 30 s over a period of 90 min at 37 °C with the medium shaking of the plate for 5 s before taking the first reading. The change in the intensity of fluorescence due to polymerization or depolymerization of tubulin was determined by setting the excitation wavelength at 360 nm and emission wavelength at 450 nm using medium gain. Reaction without any compound (DMSO, 2%) and tubulin protein served as negative controls. All experiments were performed in triplicates.
Tubulin polymerization kit
Manufactured by Cytoskeleton
Sourced in United States
The Tubulin Polymerization kit is a laboratory tool designed to study the in vitro polymerization of tubulin, a key component of the cytoskeleton. The kit provides purified tubulin and necessary reagents to monitor the polymerization process. The core function of the kit is to facilitate the analysis of tubulin dynamics for research purposes.
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Lab products found in correlation
Spectrafluor plus, by Tecan (3 mentions)
Spectramax m5e, by Molecular Devices (1 mentions)
Avance 3 spectrometer, by Bruker (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Cck 8 assay kit, by Beyotime (1 mentions)
Cell cycle analysis kit, by Beyotime (1 mentions)
Victor nivo, by PerkinElmer (1 mentions)
Infinite 200 pro fluorimeter, by Tecan (1 mentions)
11 protocols using tubulin polymerization kit
1
In vitro Tubulin Polymerization Assay
2
Tubulin Polymerization Inhibition Assay
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Tubulin polymerization kit ((#BK004) was purchased from Cytoskeleton, Inc. (Denver, CO, USA). To study the effect of the compounds on MT assembly, the absorbance-based in vitro tubulin polymerization assay was performed following the manufacturer’s protocol. The reaction mixture containing porcine tubulin (40 µM) in G-PEM buffer (80 mM PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, and 5% glycerol) supplemented with 1.0 mM GTP, in the presence of DMSO (0.18%) or the compound to be tested, was prepared and added to each well of a 96-well plate (half area, #675096, Greiner Bio One, Courtaboeuf, France). Tubulin polymerization was monitored by the increase in absorbance over time, measured by a CLARIOstarplus Microplate reader from BMG Lab technology (Champagny-sur-Marne, France). The IC50 value of each compound was estimated graphically as the concentration which decreased the maximum assembly rate of tubulin by 50% compared to the rate in the presence of DMSO (100%).
3
Microtubule Dynamics and Tubulin Polymerization
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The ability of 2-APCAs to interfere with the microtubules dynamics and tubulin polymerization was tested using the Tubulin Polymerization kit (Cytoskeleton Inc., Denver, CO, USA) as specified by the manufacturer. Results were obtained on a SpectraFluor Plus microplate reader (Tecan GmbH, Grödig Austria) and readings were taken every minute for 1 h (61 measurements in total).
4
Synthetic Methods and Biological Evaluations
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All the chemical reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA), Innochem. Ltd. (Shanghai, China) and Bide. Ltd. (Shanghai, China) and were used without further purification. Melting points were determined on an X-5 micromelting automated melting point apparatus. NMR spectra were recorded on a Bruker AvanceIII spectrometer with TMS as the internal standard. High-resolution mass spectra (HRMS) data were obtained by ThermoFisher TQ-S. The purity of the target compounds was determined by high-performance liquid chromatography (HPLC) with a TC-C18 column (4.6 mm × 250 mm, 5 μm) using the following conditions: injection volume: 10 μL; flow rate: 1 mL/min; and mobile phase: acetonitrile/water or methanol/water.
The cell lines used in this study were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). The tubulin polymerization kit was obtained from cytoskeleton, Inc. (cat.#BK011P, Denver, MA, USA). The CCK-8 assay kit, cell cycle analysis kit and Annexin V-FITC double staining kit were purchased from Beyotime Biotechnology (Shanghai, China). The fluorescent dye including Hoechst 33342, DCFH-DA and JC-1 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The cell lines used in this study were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). The tubulin polymerization kit was obtained from cytoskeleton, Inc. (cat.#BK011P, Denver, MA, USA). The CCK-8 assay kit, cell cycle analysis kit and Annexin V-FITC double staining kit were purchased from Beyotime Biotechnology (Shanghai, China). The fluorescent dye including Hoechst 33342, DCFH-DA and JC-1 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
5
Tubulin Polymerization Assay Protocol
6
Tubulin Polymerization Dynamics Assessment
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The impact of the CAs on the dynamic state of the tubulin polymerization was assessed by using the Tubulin Polymerization kit (Cytoskeleton Inc., Denver, CO, USA) as specified by the manufacturer. Results were obtained on a SpectraFluor Plus microplate reader (Tecan GmbH, Grödig, Salzburg, Austria) and readings were taken every minute for 1 h (61 measurements in total).
7
Evaluating Tubulin Inhibitors for Cancer Treatment
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Microtubules play an essential part in cellular functions, making tubulin a good target for antineoplastic drug design because of its indispensable significance. Antimitotic drugs can be anything that interferes with microtubule kinetics. It is possible to use tubulin inhibitors in conjunction with tubulin to limit the protein's stability and so impair the cell's ability to perform its normal functions. As an alternative to typical cytotoxic medications, tubulin inhibitors have been shown to be a very successful technique for eradicating cancerous tissue.31 (link) To evaluate the effect of new synthetic compounds, bovine brain tubulin (0.4 mg, 97 percent pure) and a tubulin polymerization kit (Cytoskeleton, Inc. Denver, CO) were used in this study. One hundred microliters of general tubulin buffer were used to mix the experimental compounds with the controls (colchicine, vinorelbine, and paclitaxel). In the article's ESI† details are provided.
8
Tubulin Polymerization Dynamics Assessment
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The impact of the CAs on the dynamic state of the tubulin polymerization was assessed by using the Tubulin Polymerization kit (Cytoskeleton Inc., Denver, CO, USA) as specified by the manufacturer. Results were obtained on a SpectraFluor Plus microplate reader (Tecan GmbH, Austria) and readings were taken every minute for 1 h (61 measurements in total).
9
Tubulin Polymerization Assay for Anti-Microtubule Agents
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A tubulin polymerization
kit (Cytoskeleton, Denver, CO) was used to evaluate effect of the
pyridine-linked CA-4 analogues on tubulin assembly in vitro.24 (link),25 (link) It is based on the principal that light is scattered by microtubules
to an extent that is proportional to the concentration of the microtubule
polymer. Compounds that interact with tubulin will alter the polymerization
of tubulin, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37 °C is monitored. The experimental
procedure of the assay was performed as described in version 8.2 of
the tubulin polymerization assay kit manual. Varying concentrations
of compounds were preincubated with 10 μM bovine brain tubulin
in glutamate buffer at 30 °C and then cooled to 0 °C. After
the addition of 0.4 mM GTP, the mixtures were transferred to 0 °C
cuvettes in a recording spectrophotometer and warmed to 30 °C.
Tubulin assembly was monitored by measuring the optical density at
340 nm using a BioTek Synergy 4 multifunction microplate spectrophotometer.
kit (Cytoskeleton, Denver, CO) was used to evaluate effect of the
pyridine-linked CA-4 analogues on tubulin assembly in vitro.24 (link),25 (link) It is based on the principal that light is scattered by microtubules
to an extent that is proportional to the concentration of the microtubule
polymer. Compounds that interact with tubulin will alter the polymerization
of tubulin, and this can be detected using a spectrophotometer. The
absorbance at 340 nm at 37 °C is monitored. The experimental
procedure of the assay was performed as described in version 8.2 of
the tubulin polymerization assay kit manual. Varying concentrations
of compounds were preincubated with 10 μM bovine brain tubulin
in glutamate buffer at 30 °C and then cooled to 0 °C. After
the addition of 0.4 mM GTP, the mixtures were transferred to 0 °C
cuvettes in a recording spectrophotometer and warmed to 30 °C.
Tubulin assembly was monitored by measuring the optical density at
340 nm using a BioTek Synergy 4 multifunction microplate spectrophotometer.
10
Fluorescence-Based Tubulin Polymerization Assay
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MT polymerization assays were performed using a fluorescence-based Tubulin Polymerization kit (Cytoskeleton Inc, Denver CO, Cat. # BK011P) following the manufacturer’s instructions.
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