Product Uses Include
- Economical alternative to T240 in primary screens for tubulin ligand drugs
- High-throughput tubulin polymerization or depolymerization screens
- Production of microtubule substrates for HTS motor assays
MaterialTubulin protein has been isolated from porcine brain. The final product contains approximately 97% tubulin and 3% Microtubule Associated Proteins (MAPs). This version of porcine tubulin is an economical alternative to our highly purified tubulin (Cat. # T240) for anti-tubulin ligand drug discovery. It has been formulated to be compatible with 96 or 384-well format polymerization assays such as in Biochem™ kits BK004P or BK011P .
PurityProtein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS-PAGE gel. HTS02 contains 97% tubulin (MW. 55 kDa) and 3% MAPs (MW 35-280 kDa).
Figure 1: A 100 µg sample of HTS03 protein was separated by electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).
Biological ActivityAn ASSAY UNIT is defined as the amount of HTS03 protein needed to achieve a tubulin polymerization signal of OD340 of 0.1 - 0.15 in 30 minutes at 37°C in G-PEM buffer. The assay volume is 100 µl and assumes a spectrophotometer pathlength of 0.5 cm. The protein concentration for HTS02 in this assay is approximately 4 mg/ml. The biological activity of HTS03 is assessed by a tubulin polymerization assay. One ASSAY UNIT of tubulin is used for each polymeriztion assay. An OD340 of 0.10 - 0.15 is required to pass quality control. Tubulin polymerization must also be responsive to polymerization enhancers (paclitaxel) and inhibitors (nocodazole) at 5 µM drug concentration.
Figure 2: Tubulin polymerization in a 96-well format using HTS03. All samples are in duplicates. Each well has a mini-polymerization curve with 20 min on the x-axis and 0.30 OD 340 nm on the y-axis. The Vmax parameter is used to compare inhibitors, the CV is 13% in this format, so a 50% cut off is required for a 98% confidence.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Li et al., 2012. Orally Bioavailable Tubulin Antagonists for Paclitaxel-Refractory Cancer. Pharm. Res. DOI: 10.1007/s11095-012-0814-5.
Also, see references for Cat. # BK004P which uses HTS03 for the tubulin source
Question 1: Does tubulin polymerization using 97% pure porcine brain tubulin (Cat. # HTS03) require additional enhancers such as glycerol or taxol?
Answer 1: No additional enhancers are required when using 3 or 4 mg/ml of the MAP-enriched 97% pure tubulin. The recommended polymerization reaction using 97% pure tubulin contains 100 μl of 3 or 4 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 will reach a maximal OD340 between 0.15 – 0.25 within 30 minutes. In this experimental set up (100 μl volume in a spectrophotometer with a pathlength of 0.5 cm), an OD340 of 0.1 is approximately equal to 1 mg per ml of tubulin polymer mass. Under these conditions only 40% of the tubulin is polymerized, offering the flexibity to study how polymerization enhancers (e.g., taxol) or inhibitors (e.g., nocodazole) alter tubulin polymerization. For enhancers, we recommend using 3 mg/ml tubulin whereas for inhibitors, 4 mg/ml tubulin works better.
Question 2: Upon resuspension as directed, what are the components of the final buffer?
Answer 2: Upon reconstitution as directed, the tubulin will be in the following buffer: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 7.0, 1 mM GTP, 1% Ficoll, 5% sucrose and <1mM DTT.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
Cytoskeleton公司成立于1993年，专注于生物化学和细胞过程研究中的纯化蛋白和便捷试剂盒开发与生产。公司提供药物筛选、信号转导、细胞骨架研究相关的系列试剂盒和产品，尤其以细胞骨架相关研究见长，既能满足于样品较少的科学研究，也可以用于小规模筛选研究和高通量大规模筛选研究。此外，公司还提供微管蛋白，肌动蛋白，小G蛋白，GAPs，GEFs等现有产品的药物筛选服务。
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