Product Uses Include
Tubulin and microtubule associated proteins (MAPs) has been purified from porcine brain by an adaptation of the method of Shelanski et al. (1). Tubulin is supplied as a white lyophilized powder. The protein composition is approximately 70% tubulin (55 kDa heterodimer) and 30% MAPs. The MAPs in this product act to stabilize microtubules and to enhance tubulin polymerization. MAP rich tubulin can polymerize efficiently at 1-2 mg/ml.
MAP-rich tubulin is also available from a bovine source (Cat. # ML113).
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are approximately 70% tubulin and 30% MAPs.
One unit of MAP-rich tubulin is defined as 5.0 mg of protein (as determined by the Precision Red Protein Assay Reagent, Cat. # ADV02). MAP-rich tubulin will polymerize efficiently at protein concentrations above 1mg/ml and temperatures above 24°C.
N.-J. Chiang et al. 2013. A novel synthetic microtubule inhibitor, MPT0B214 exhibits antitumor activity in human tumor cells through mitochondria-dependent intrinsic pathway. PLoS ONE. 8, e58953.
S. Rath et al. 2012. Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives. Br. J. Pharmacol. 167, 1048-1061.
C.-C. Hsieh et al. 2010. Chamaecypanone C, a novel skeleton microtubule inhibitor, with anticancer activity by trigger caspase 8-Fas/FasL dependent apoptotic pathway in human cancer cells. Biochem. Pharmacol. 79, 1261-1271.
Question 1: What is the difference between 70% pure porcine brain tubulin (Cat. # ML116) and 70% pure bovine brain tubulin (Cat. # ML113)?
Answer 1: The only difference is the source of the tubulin: bovine vs porcine brains. Functionally, there are no significant differences between tubulin isolated from bovine brains vs porcine brains. Stringent quality control and in-house testing has revealed no differences in polymerization, response to drugs (enhancers and inhibitors) or binding to accessory proteins (e.g., motor proteins). (See Comparison of Porcine and Bovine tubulin)
Question 2: Does tubulin polymerization using 70% pure porcine brain tubulin/30% MAPs (Cat. # ML116) require additional enhancers such as glycerol or taxol?
Answer 2: No additional enhancers are required when using 2 mg/ml of the MAP-enriched 70% pure tubulin. The recommended polymerization reaction using 70% pure tubulin contains 180 μl of 2 mg/ml tubulin in 80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCl2 and 1 mM GTP. Polymerization is started by incubation at 37°C and followed by absorption readings at 340 nm. Under these conditions polymerization reaches a maximal OD340 between 0.3 - 0.4 within 20 minutes. In this experimental set up (180 μl volume in a spectrophotometer with a pathlength of 0.8 cm using Corning Costar’s half area well plate Cat. # 3696) an OD340 of 0.16 is approximately equal to 1 mg per ml of tubulin polymer mass. Thus, under the conditions described, approximately 100% of the tubulin is polymerized. If polymerization enhancers such as taxol are to be used or tested, we recommend reducing the tubulin concentration to 1 mg/ml. These conditions will result in a more pronounced nucleation phase and a lower polymerization curve.
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