Tubulin protein: MAP rich: bovine brain
Product Uses Include
- Studying the effects of MAPs on microtubule dynamics
- Studying the binding sites between MAPs and tubulin.
- Studying the effects of anti-microtubule drugs on microtubule polymerization.
Tubulin and microtubule associated proteins (MAPs) has been purified from bovine 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 (see Figure 1). 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 porcine source (Cat. # ML116)
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are approximately 70% tubulin and 30% MAPs.
|Figure 1: 100 µg of ML113 was run on a 10% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02).||
One unit of ML113 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 1 mg/ml and temperatures above 24°C.
|Figure 2: MAP-rich tubulin polymerization under different conditions. Samples of ML113 in G-PEM buffer were placed in 96-well plates and incubated at the specified temperatures. Sample 1: 2 mg/ml at 37°C. Sample 2: 2 mg/ml at 24°C. Sample 3: 1 mg/ml at 24°C. Sample 4, same as 3 except in the presence of 3 µM nocodazole. Tubulin polymerization was observed by measuring the optical density at 340 nm over time.||
Shelanski M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768
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Kuo, C. C., Hsieh, H. P., Pan, W. Y., Chen, C. P., Liou, J. P., Lee, S. J., Chang, Y. L., Chen, L. T., Chen, C. T. and Chang, J. Y. (2004). BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res. 64, 4621-4628.
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Question 1: What is the difference between 70% pure bovine brain tubulin (Cat. # ML113) and 70% pure porcine brain tubulin (Cat. # ML116)?
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 bovine brain tubulin/30% MAPs (Cat. # ML113) 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, >90% 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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