Gmpcpp

Manufactured by Jena Biosciences

Sourced in Germany

GMPCPP is a non-hydrolyzable GTP analog that can be used to study microtubule dynamics in vitro. It binds to and stabilizes microtubules, preventing their disassembly.

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54 protocols using gmpcpp

Biotinylated GMPCPP-stabilized Microtubule Preparation

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Short biotinylated GMPCPP-stabilized microtubules were polymerized from a mixture of Alexa647-tubulin (12.1 μM, labeling ratio 0.1) and biotin-tubulin (6 μM) in the presence of 0.5 μM GMPCPP (Jena Bioscience) in 60 μl of BRB80 (80 mM PIPES, 1 mM MgCl₂, 1 mM EGTA) for 1 h at 37°C, centrifuged at 17,000 x g for 15 min, washed with warm BRB80 (37°C), centrifuged again at 17,000 x g for 10 min, resuspended in 50 μl BRB80 and kept at room temperature.
Long biotinylated GMPCPP-stabilized microtubules were polymerized from a mixture of Alexa647-tubulin (1.7 μM, labeling ratio 0.1) and biotin-tubulin (0.5 μM) in the presence of 0.3 μM GMPCPP (Jena Bioscience) in 300 μl of BRB80 for 2 h at 37°C, centrifuged at 17,000 x g for 15 min, washed with warm BRB80 (37°C), centrifuged again at 17,000 x g for 10 min, resuspended in 30 μl BRB80 and kept at room temperature.

Hannabuss J., Lera-Ramirez M., Cade N.I., Fourniol F.J., Nédélec F, & Surrey T. (2019). Self-Organization of Minimal Anaphase Spindle Midzone Bundles. Current Biology, 29(13), 2120-2130.e7.

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Microtubule Stabilization and Polymerization

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To obtain taxol-stabilized microtubules, tubulin at a concentration of 4.4 mg/ml was polymerized for 30 min at 37 °C in BRB80 buffer supplemented with 4.8% DMSO, 4 mM MgCl₂, and 1 mM GTP, after which BRB80 with 20 µM paclitaxel was added. For GMPCPP-stabilized microtubules, tubulin at a concentration of 0.25 mg/ml was incubated for 2 h at 37 °C in BRB80 buffer supplemented with 1 mM MgCl₂ and 1 mM GMPCPP (Jena Bioscience, Jena, Germany). Dolastatin-10 rings resulted from the polymerization of 2.2 mg/ml tubulin for 40 min at room temperature with 40 µM dolastatin-10 in 80 mM PIPES (pH 6.9), 50 mM KCl, 1 mM EGTA, 1 mM MgCl₂, and 1 mM DTT. After the polymerization, all abovementioned polymers were pelleted at 30,000 × g for 40 min at 37 °C and the pellet was suspended in warm BRB80 with 20 µM taxol to the required concentration. Zn-sheets were prepared via 2 h incubation of 3 mg/ml tubulin at 37 °C in 80 mM MES, 200 mM NaCl, 3 mM GTP, 1.25 mM MgSO₄, 1.25 mM ZnSO₄, and 0.025 mg/ml pepstatin, adjusted to a pH of 5.5 with NaOH. Zn-sheets were then stabilized by the addition of paclitaxel to a final concentration of 31.5 µM. Zn-sheets were pelleted at 30,000 × g for 40 min at 37 °C and the pellet was suspended in a warm solution of 80 mM MES, 200 mM NaCl, 1.25 mM MgSO₄, and 20 µM taxol, adjusted to a of pH 5.5 with NaOH.

Skultetyova L., Ustinova K., Kutil Z., Novakova Z., Pavlicek J., Mikesova J., Trapl D., Baranova P., Havlinova B., Hubalek M., Lansky Z, & Barinka C. (2017). Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules. Scientific Reports, 7, 11547.

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Preparation of Taxol and GMP-CPP Stabilized Microtubules

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Taxol-stabilized microtubules were grown at 37°C for 1 h from 30 µM Atto647N-labeled pig brain tubulin (labeled:unlabeled = 1:20) in BRB80, pH 6.8, supplemented with 4 mM MgCl₂, 1 mM GTP (Jena Bioscience GmbH, Jena, Germany), and 4.8% (vol/vol) dimethyl sulfoxide (DMSO). Microtubules were sedimented at room temperature for 30 min at 17,000 × g and resuspended in warm (i.e., 37ºC) BRB80 supplemented with 10 µM Taxol (Sigma-Aldrich, Hamburg, Germany). Microtubules were stored in the dark at room temperature and kept for a maximal 48 h. GMP-CPP-stabilized microtubule were grown 37°C for 2 h from 2 µM Atto647N-labeled pig brain tubulin (labeled:unlabeled = 1:4 [bright] or 1:20 [dim]) in BRB80, 1 mM GMP-CPP (Jena Bioscience). Microtubules were sedimented at room temperature for 30 min at 17,000 × g and resuspended in warm BRB80. For subtilisin treatment, GMP-CPP microtubules were polymerized and sedimented as above and resuspended in BRB80 supplemented with 10 µm Taxol and 200 µg/ml subtilisin A (Sigma-Aldrich). Microtubules were incubated for 1 h at 30°C before protease digest was stopped by the addition of 10 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich). Microtubules were sedimented as above and resuspended in warm BRB80. The samples were split and analyzed on a 4–12/% precast SDS–PAGE gel (ThermoFisher, Langenselbold, Germany) or directly used in the TIRF assay.

Drechsler H., Xu Y., Geyer V.F., Zhang Y, & Diez S. (2019). Multivalent electrostatic microtubule interactions of synthetic peptides are sufficient to mimic advanced MAP-like behavior. Molecular Biology of the Cell, 30(24), 2953-2968.

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Imaging Kif5c-GFP on GMPCPP-stabilized MTs

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GMPCPP-MTs were polymerized using a mixture of 20 μM tubulin extracted from mouse brain, 1 mM GMPCPP (Jena Bioscience; catalog no.: NU-405L) and 4 mM MgCl₂ in BRB80, which was incubated for 2 h at 37 ˚C. Polymerized MTs were collected using an air-driven ultracentrifuge (Beckman; catalog no.: 340401), which were then resuspended in BRB80 with 20 μM taxol (Cell Signaling Technology; catalog no.: 9807), and stored at 37 ˚C. Part of MTs was treated with TTLL4 or TTLL7 for 2 h at room temperature of 37 °C. Then, MTs were immobilized on the surface of a cover glass using the tubulin antibody links (Sigma; catalog no.: T7816). Next, Kif5c-GFP was added into the flow cell in the imaging buffer (BRB80 supplemented with 1 mM ATP, 20 μM taxol, 80 mM d-glucose, 0.4 mg/ml glucose oxidase, 0.2 mg/ml catalase, 0.8 mg/ml casein, 1% β-mercaptoethanol, and 0.001% Tween-20). Images were recorded every 100 ms with a 50 ms exposure. The sample was kept at 35 °C using a temperature controller (Tokai Hit). Images were recorded using a TIRFM (Olympus).

Zhang X., Li X., Chen W., Wang Y., Diao L., Gao Y., Wang H., Bao L., Liang X, & Wu H.Y. (2023). The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin. The Journal of Biological Chemistry, 299(7), 104923.

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GMPCPP-stabilized microtubule construction

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For the construction of GMPCPP-stabilized α1A/β2A and α1C/β2A microtubules, 15‒20 µM α1A/β2A or α1C/β2A tubulins were incubated with 1 mM GMPCPP (Jena Biosciences) at 37°C for 1 h. To remove unpolymerized tubulin, the solution was centrifuged at 126000 g for 5 min at 27°C and the supernatant was discarded. Then, the microtubule pellet was resuspended in cold BRB80 and depolymerized at 4°C for 20 min, followed by a second round of polymerization at 37°C with 1 mM GMPCPP for 1 h. Microtubule seeds were pelleted as above and resuspended in 37°C pre-warmed BRB80 with ∼80% volume of initial reaction. The microtubule solution was diluted at a ratio of 1:2‒1:3, and 2.2 µl was applied to the plasma-cleaned grid using a Vitrobot Mark IV (Thermo Fisher Scientific). Images were collected using a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV. All images were recorded on a K2 Summit direct electron detector (Gatan) at a nominal magnification of 18000×, yielding a pixel size of 1.3 Å.

Diao L., Liu M.Y., Song Y.L., Zhang X., Liang X, & Bao L. (2021). α1A and α1C form microtubules to display distinct properties mainly mediated by their C-terminal tails. Journal of Molecular Cell Biology, 13(12), 864-875.

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Preparation of Fluorescent Microtubules

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Unlabelled porcine tubulin, Hilyte488-tubulin and biotin-tubulin were supplied by Cytoskeleton Inc. (Denver, CO). To polymerise fluorescent GMPCPP-stabilised microtubules, 1.66 μM unlabelled tubulin, 0.15 μM Hilyte488-tubulin and 0.4 μM biotin-tubulin were incubated together with 0.5 mM GMPCPP (Jena Bioscience (Jena, Germany)) in BRB80 (80 mM PIPES pH 6.85, 2 mM MgCl₂, 0.5 mM EGTA) for 2 hr 30 min at 37°C. Polymerised microtubules were sedimented by centrifugation at 18,400 x g in an Eppendorf 5424 centrifuge for 8 min at room temperature. The microtubule pellet was washed by resuspension in 1 ml of prewarmed BRB80 and sedimented again by centrifugation. Microtubules were then resuspended in BRB80 that had been prewarmed to 37°C and kept at room temperature for use within 1–2 hr.

Baumbach J., Murthy A., McClintock M.A., Dix C.I., Zalyte R., Hoang H.T, & Bullock S.L. (2017). Lissencephaly-1 is a context-dependent regulator of the human dynein complex. eLife, 6, e21768.

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GTPase Assays and Tryptophan Emission in FtsZ

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High speed pelleting and co-pelleting, and GTPase assays were performed in HEK50 buffer (50 mM HEPES-KOH pH 7.2, 50 mM KCl, 0.1 mM EDTA) as described previously (Goley et al., 2010 (link); Sundararajan et al., 2015 (link)). When indicated, MgCl₂ was added to 1, 2.5 or 10 mM, GTP was added to 2 mM, or GMP-CPP (Jena Bioscience) was added to 0.2 mM. Tryptophan assay was performed by measuring tryptophan emission at 344 nm of FtsZ L72W ± FtsZ in HEK50 buffer with ZapA or ZauP at after exciting at 295 nm using Fluoromax-3 as described (Milam and Erickson, 2013 (link)).

Woldemeskel S., McQuillen R., Hessel A.M., Xiao J, & Goley E.D. (2017). A Conserved Coiled-Coil Protein Pair Focuses the Cytokinetic Z-Ring in Caulobacter crescentus. Molecular microbiology, 105(5), 721-740.

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Fluorescent-labelled Microtubule Preparation

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Microtubules were polymerised from porcine tubulin (Cytoskeleton Inc., Denver, CO) and labelled with fluorophores and biotin by stochastic incorporation of labelled dimers into the microtubule lattice. Mixes of 1.66 μM unlabelled tubulin, 0.15 μM Hilyte488-tubulin, and 0.4 μM biotin-tubulin were incubated in BRB80 (80 mM PIPES pH 6.85, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT) with 0.5 mM GMPCPP (Jena Bioscience, Jena, Germany) for 2–4 hr at 37°C. Polymerised microtubules were pelleted in a room temperature table top centrifuge at 18,400 x g for 8.5 min, and washed once with pre-warmed (37°C) BRB80. After pelleting once more, the microtubules were gently resuspended in pre-warmed (37°C) BRB80 containing 40 μM paclitaxel (taxol; Sigma-Aldrich) and used on the same day.

McClintock M.A., Dix C.I., Johnson C.M., McLaughlin S.H., Maizels R.J., Hoang H.T, & Bullock S.L. (2018). RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs. eLife, 7, e36312.

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Polarity-marked Microtubule Preparation

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All taxol-stabilized polarity-marked microtubules (tetramethylrhodamine (TMR), Alexa 488, and HiLyte 647) with bright plus ends were prepared as previously described64 . To make the polarity-marked microtubules, a dim tubulin mix (containing 17 μM unlabelled tubulin and 0.8 μM fluorescently labelled tubulin) was first incubated in BRB80 (80 mM PIPES, pH 6.8, 1 mM EGTA and 1 mM MgCl₂) with 0.5 mM guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP) (Jena Bioscience) at 37 °C for 2 h to make dim microtubules, and then centrifuged at 250,000 g for 7 min at 37 °C in a TLA100 rotor (Beckman). The pellet was resuspended in a bright tubulin mix (containing 7.5 μM unlabelled tubulin, 4 μM fluorescently labelled tubulin and 15 μM N-ethylmaleimide-tubulin) in BRB80 with 0.2 mM GMPCPP and incubated at 37 °C for additional 15 min to cap the plus ends. The resulting polarity-marked microtubules were pelleted at 20,000 g for 7 min at 37 °C in the TLA100 rotor and finally resuspended in BRB80 with 40 μM taxol. For making track microtubules used in single-molecule assays and microtubule-sliding assays, the dim tubulin mix also included additional 17 μM biotinylated tubulin.

Popchock A.R., Tseng K.F., Wang P., Karplus P.A., Xiang X, & Qiu W. (2017). The mitotic kinesin-14 KlpA contains a context-dependent directionality switch. Nature Communications, 8, 13999.

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Fluorescent Labeling of Tubulin for Microtubule Stabilization

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Bovine brain tubulin was labeled following previously described methods (Hyman et al., 1991 (link)). Using Cy5-NHS ester (GE Healthcare, PA15101) yielded 54–70% labeling. Using Alexa-568 NHS ester (Invitrogen, A20003) yielded 36–40% labeling. Labeling efficiency with biotin-PEG4-NHS (Thermo Scientific, A39259) was not calculated.
Single-cycled GMPCPP-stabilized microtubules were made as previously described (Gell et al., 2010 (link)). Briefly, 12 μM unlabeled tubulin + 1 μM Alexa-568 tubulin + 1 μM biotin tubulin was polymerized in BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl₂) in the presence of 1 mM GMPCPP (Jena Bioscience, NU-405L) for 1 hr at 37°C. For GMPCPP-stabilized microtubules without any labels, 14 μM unlabeled tubulin was polymerized. For GMPCPP-stabilized microtubules without biotin, 13 μM unlabeled tubulin + 1 μM Alexa-568 tubulin was polymerized.

Alfaro-Aco R., Thawani A, & Petry S. (2020). Biochemical reconstitution of branching microtubule nucleation. eLife, 9, e49797.

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