S-methyl DM1 and other maytansinoids were synthesized as described previously [6 (link)]. 3[H]-S-methyl DM1 was synthesized at American Radiolabeled Chemicals. 1,2-dioleoyl-sn-glycero-3-phosphocholine in chloroform (Avanti Polar Lipids) was stored under argon at-20o C. 3[H]-Paclitaxel was from Moravek Biochemicals. MCF7 cell tubulin and bovine brain tubulin (>99%; isolated by the method described in [10 (link)]) were purchased from Cytoskeleton, Inc. Paclitaxel was from Tocris; nocodazole, vinblastine sulfate, demecolcine, monoclonal anti-α-tubulin clone B-5–1–2 murine IgG1 T6074, RIPA buffer from Sigma; Halt Protease & Phospatase Inhibitor Cocktail (no EDTA or other chelators) was from Thermo Scientific; DilC18(3) and MagicMark XP protein size marker were purchased from Invitrogen; peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) 115–036–062 was purchased from Jackson Labs; ECL Advanced Blocking Agent CPK1075 was purchased from GE Healthcare.
Bovine brain tubulin
Manufactured by Cytoskeleton
Sourced in United States
Bovine brain tubulin is a purified protein isolated from bovine brain tissue. It is a key structural component of the cytoskeleton in eukaryotic cells, playing a crucial role in cellular processes such as cell division, cell motility, and intracellular transport.
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Lab products found in correlation
Synergy 4 microplate reader, by Agilent Technologies (2 mentions)
Vinblastine sulfate, by Merck Group (1 mentions)
Demecolcine, by Merck Group (1 mentions)
Nocodazole, by Bio-Techne (1 mentions)
Paclitaxel, by Bio-Techne (1 mentions)
Peroxidase conjugated affinipure f ab 2 fragment goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
3h paclitaxel, by Moravek Biochemicals (1 mentions)
3h vinblastine, by PerkinElmer (1 mentions)
Docetaxel, by Sanofi (1 mentions)
Gen5 data analysis software, by Agilent Technologies (1 mentions)
Dioleoylphosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
Dioleoylphosphatidylcholine dopc, by Avanti Polar Lipids (1 mentions)
Diphytanoylphosphatidylcholine dphpc, by Avanti Polar Lipids (1 mentions)
Vitrobot 4, by Thermo Fisher Scientific (1 mentions)
7 protocols using bovine brain tubulin
1
Synthesis and Characterization of Maytansinoid Compounds
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S-methyl DM1 and other maytansinoids were synthesized as described previously [6 (link)]. 3[H]-S-methyl DM1 was synthesized at American Radiolabeled Chemicals. 1,2-dioleoyl-sn-glycero-3-phosphocholine in chloroform (Avanti Polar Lipids) was stored under argon at-20o C. 3[H]-Paclitaxel was from Moravek Biochemicals. MCF7 cell tubulin and bovine brain tubulin (>99%; isolated by the method described in [10 (link)]) were purchased from Cytoskeleton, Inc. Paclitaxel was from Tocris; nocodazole, vinblastine sulfate, demecolcine, monoclonal anti-α-tubulin clone B-5–1–2 murine IgG1 T6074, RIPA buffer from Sigma; Halt Protease & Phospatase Inhibitor Cocktail (no EDTA or other chelators) was from Thermo Scientific; DilC18(3) and MagicMark XP protein size marker were purchased from Invitrogen; peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) 115–036–062 was purchased from Jackson Labs; ECL Advanced Blocking Agent CPK1075 was purchased from GE Healthcare.
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S-methyl DM1 and other maytansinoids were synthesized as described previously [6 (link)]. 3[H]-S-methyl DM1 was synthesized at American Radiolabeled Chemicals. 1,2-dioleoyl-sn-glycero-3-phosphocholine in chloroform (Avanti Polar Lipids) was stored under argon at-20o C. 3[H]-Paclitaxel was from Moravek Biochemicals. MCF7 cell tubulin and bovine brain tubulin (>99%; isolated by the method described in [10 (link)]) were purchased from Cytoskeleton, Inc. Paclitaxel was from Tocris; nocodazole, vinblastine sulfate, demecolcine, monoclonal anti-α-tubulin clone B-5–1–2 murine IgG1 T6074, RIPA buffer from Sigma; Halt Protease & Phospatase Inhibitor Cocktail (no EDTA or other chelators) was from Thermo Scientific; DilC18(3) and MagicMark XP protein size marker were purchased from Invitrogen; peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti mouse IgG (H+L) 115–036–062 was purchased from Jackson Labs; ECL Advanced Blocking Agent CPK1075 was purchased from GE Healthcare.
2
Tubulin Polymerization Assay
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Bovine brain tubulin (0.4
mg, >97% pure) (Cytoskeleton, Denver, CO)
was mixed with 10 μM of the test compounds and incubated in
100 μL of general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP) at pH 6.9. The absorbance of
wavelength at 340 nm was monitored every 1 min for 20 min by the SYNERGY
4 Microplate Reader (Bio-Tek Instruments, Winooski, VT). The spectrophotometer
was set at 37 °C for tubulin polymerization.
mg, >97% pure) (Cytoskeleton, Denver, CO)
was mixed with 10 μM of the test compounds and incubated in
100 μL of general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP) at pH 6.9. The absorbance of
wavelength at 340 nm was monitored every 1 min for 20 min by the SYNERGY
4 Microplate Reader (Bio-Tek Instruments, Winooski, VT). The spectrophotometer
was set at 37 °C for tubulin polymerization.
3
Recombinant LRRK2 and Tau Purification
4
Taxol and Docetaxel Tubulin Binding Assays
5
Tubulin Polymerization Kinetics Assay
6
Lipid Membrane Protein Interactions
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Dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), diphytanoylphosphatidylcholine (DPhPC), and dierucoylphosphatidylcholine (di(C22:1)PC) were purchased from Avanti Polar Lipids, Inc. Alabaster, AL. Gramicidin A (grA) was generously gifted from O. S. Andersen, Cornell University Medical College. Bovine brain tubulin was obtained from Cytoskeleton (Denver, CO, USA). The peptide corresponding to the α-tubulin H10 domain sequence (residues 328–346), VNAAIATIKTKRSIQFVDW, was synthesized in an FDA/CBER peptide synthesis core (White Oak, MD, USA) and dissolved in DMSO. All other chemicals were analytical grade.
7
Microtubule Polymerization and Motor Protein Binding
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MTs were polymerized from bovine brain tubulin (Cytoskeleton, Inc.), at a final concentration of 5 mg/mL, at 37 °C for 1.5 hr in 100 mM MES pH 6.5, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT and 5 mM GTP buffer. MTs were stabilized with 1 mM paclitaxel (Calbiochem) in DMSO with incubation for a further 1.5 hr at 37 °C. 100 μM of each motor domain construct was mixed with 14 μM stabilized MTs, and 4 μl of this mixture was applied onto Quantifoil R 2/2 holey carbon grids glow-discharged in air, which were blotted and plunge frozen into liquid ethane using a Vitrobot IV (FEI/Thermo Fisher) operating at room temperature and 100% humidity.
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