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.
Porcine tubulin
Manufactured by Cytoskeleton
Sourced in United States
Porcine tubulin is a purified protein derived from pig brain tissue. It is a key structural component of the cytoskeleton, playing a crucial role in cell division, intracellular transport, and maintaining cell shape. Porcine tubulin can be used for a variety of research applications in cell biology and biochemistry.
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Lab products found in correlation
Clarity reagent, by Bio-Rad (2 mentions)
Fluor s device, by Bio-Rad (2 mentions)
Gmpcpp, by Jena Biosciences (1 mentions)
Paclitaxel taxol, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Casein, by Merck Group (1 mentions)
Mouse albumin, by Innovative Research (1 mentions)
Taxol, by Merck Group (1 mentions)
Proteoblock protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Sirt2 activity assay kit, by Abcam (1 mentions)
B 5 1 2, by Abcam (1 mentions)
Clariostar plus, by BMG Labtech (1 mentions)
Ab193468, by Abcam (1 mentions)
Adi sab 300 j, by Enzo Life Sciences (1 mentions)
Adi sab 100 j, by Enzo Life Sciences (1 mentions)
Tri carb 4910 tr, by PerkinElmer (1 mentions)
14 protocols using porcine tubulin
1
Fluorescent-labelled Microtubule Preparation
2
Mms19-Tubulin Interaction Assay
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50μg of purified Mms19 (Tagged with 5X Histidine at C-terminus, synthesized by Genscript Inc and solubilized in 20mM Tris, 150mM NaCl, 0.5 M Arginine) was incubated with 50μg purified porcine tubulin (Purchased from Cytoskeleton Inc) at 4°C for 2 hrs. This mixture was subsequently incubated with Ni-NTA agarose (equilibrated with 20mM Tris-Cl and 250mM NaCl) for 1 hr at 4°C. The beads were then washed three times with wash buffer containing 20mM Tris, 250mM NaCl, and 20mM Imidazole, and the bound proteins were eluted by adding elution buffer (20mM Tris, 250mM NaCl, and 500mM Imidazole) to the resin on ice for 10 min. The eluate was then analyzed by probing Western blots with anti-Mms19 antibodies and rabbit anti-alpha-Tubulin antibodies.
3
Comprehensive Protein Reagent Database for Immunoassay Development
4
Cryo-EM Visualization of Cin8-Microtubule Interactions
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The MT polymerization reaction mixture contained final concentrations of porcine tubulin (10 mg/ml; Cytoskeleton Inc.) and 1.5 mM GTP in general tubulin buffer (GTB; Cytoskeleton Inc.). The mixture was incubated for 50 min at 37°C, followed by the addition of Taxol (Sigma-Aldrich) to a final concentration of 100 μM, and another 20-min incubation at 37°C. After the polymerization of the MTs, the mixture was diluted ninefold with GTB supplemented with 10 μM Taxol (GTB-Tx), and centrifuged for 20 min at 15,000g. The supernatant was discarded, and the pellet was resuspended in 40 μl of GTB-Tx. A pre-reaction mixture was then prepared so as to provide final concentrations of 0.1 mM AMPPNP and 100 mM KCl in GTB-Tx. The Cin8L8Kip1 construct was added in a ratio of 4:1 to the MTs (tubulin concentration of the MTs was ~0.13 mg/ml). The mixture was mixed gently and incubated for 10 min. Cryo-EM samples were prepared by plunge-freezing into liquid ethane. Thereafter, 3 μl of the mixture was applied onto glow-discharged Lacey F/C grids (01883-F), blotted for 20 s, and then vitrified by rapidly plunging into liquid ethane using an FEI Vitrobot operating at room temperature and 100% humidity. The frozen samples were stored in liquid nitrogen until imaging.
5
Preparation of Taxol-Stabilized Microtubules
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Porcine tubulin (Cytoskeleton, Denver, CO) was polymerized according to the manufacturer’s protocol to produce MTs with a tubulin concentration of 140 μM. All MTs used in this work were taxol-stabilized. Polymerized MTs are then diluted 100x in MT buffer (35 mM PIPES, 5 mM MgSO4 pH 7.2, supplanted with 1 mM GTP, and 40 μM Taxol.)
6
Deacetylation Assay of Tubulin
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For deacetylation assays 0.5 μg porcine tubulin (Cytoskeleton Inc.) was incubated in 50 mM Tris-HCl, pH 8.0, 10% (v/v) Glycerol, 1 mM DTT, 0.1 mM EDTA containing ProteoBlock protease inhibitor cocktail (Fermentas) with 1 μg of GST or GST fusion proteins. Tubulin (B-5-1-2, Abcam) and acetylated tubulin (K40ac antibody 6-11B-1, Sigma-Aldrich) were detected on Western Blots as described before [15 (link)]. Moreover we employed the SIRT2 activity assay kit (ab156066, Abcam, Cambridge, UK) according to the manufacturer’s protocol using GST-tagged fusion proteins (0.5 μg/assay).
7
Tubulin Polymerization Assay Protocol
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Tubulin polymerization was assessed with the use of purified porcine tubulin purchased from Cytoskeleton Inc. (Denver, CO, USA) in accordance with a protocol recommended by the manufacturer. Tubulin was dissolved in a buffer containing: 80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM EGTA and 1 mM GTP, at a final concentration of 3 mg/mL and placed in a 96-well plate (0.3 mg per well). The polymerization reaction was started by increasing temperature from 4 to 37 °C upon transfer of the reaction mixture to a pre-warmed plate. The assembly of microtubules was monitored spectrophotometrically by measuring absorbance at 350 nm for 60 min, at a temperature of 37 °C. Paclitaxel was used as stabilizing positive control.
8
Quantifying CCP-Mediated Tubulin Deglutamylation
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To determine CCP activity towards polyglutamated tubulin, a 20 μl reaction mixture containing 1 μg of respective purified CCP5 and/or Nna1 and 2 μg porcine tubulin (Cytoskeleton Inc., Denver, USA) in PBS was incubated at 37 °C for 1 h. Reactions containing heat denatured enzyme served as controls of specificity. To detect the enzyme activities of CCP5 splicing variants, HEK293 cells were transfected with respective variants harbored in the pCMV-myc vector to generate N-terminal tagged proteins. 40 h after transfection, cells were washed with pre-chilled PBS and lysed in PBS containing 0.2% NP-40. Cell lysate was centrifuged at 40,000 × g at 4 °C for 20 min. 20 μl of the supernatant was incubated with 1 μg porcine tubulin at 37 °C for 5 h. The lysate from cells transfected with myc-tagged LacZ or supplementing 5 mM of the metallocarboxypeptidase inhibitor, 1,10-phenanthroline (OP) to inactivate the enzyme served as controls42 (link)43 (link). Reactions were terminated with 3x sample buffer and denatured at 95 °C. Subsequently samples were subjected to immunoblot analysis using GT335 (anti-branching point glutamate), polyE (anti-polyglutamate), EP1332Y (anti-α-tubulin), or mouse anti-myc antibodies.
9
Tubulin Polymerization Inhibition Assay
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Porcine tubulin (20 µM, Cytoskeleton, Inc., T240; Denver, CO, USA) was incubated with compounds for 45 min at 37 °C in 80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 0.2 mM GTP prior to the addition of colchicine for a 10 µM final concentration (Selleckchem S2284; Houston, TX, USA). After a 45 min incubation, the fluorescence of the colchicine–tubulin complex was measured at EX 380/EM 435 nm with the CLARIOstar Plus plate reader (BMG Labtech; Ortenberg, Germany).
10
Immuno-purification and Western Blot Analysis
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This method was applied to the immuno-purified CalS complex. Proteins were denatured in LSB and separated by electrophoresis as described above; proteins were transferred to membranes and then underwent a process of denaturation/renaturation according to a protocol based on decreasing concentrations of guanidine-HCl (Yuliang et al. 2007 (link)). After overnight blocking, membranes were incubated with porcine tubulin (Cytoskeleton Inc.) at a concentration of 1 mg/mL in TBS supplemented with 0.1% Tween-20 (TBST) for 2 h at room temperature. After washing in TBS, membranes were incubated with primary anti-α-tubulin antibody B-5–1-2 (mouse) diluted 1:5000 in TBST or with antibody HDA (rabbit) against CalS diluted 1:300 in TBST. After a 1-h incubation at room temperature, membranes were washed in TBS and then incubated with secondary antibodies against mouse or rabbit immunoglobulins conjugated with HRP. Antibodies were used at a dilution of 1:5000 in TBST for 1 h at room temperature. After further washing in TBS, membranes were processed with the Clarity reagent (Bio-Rad) and the signal was detected with the Fluor-S device (Bio-Rad).
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