Important Notice:
This product is no longer available. Cytoskeleton recommends using Tubulin protein (97% pure): Porcine brain (Cat. # HTS03) as a replacment.
Product Uses Include
Material
Tubulin protein has been isolated from bovine brain. The final product contains approximately 97% tubulin and 3% Microtubule Associated Proteins (MAPs). This version of bovine tubulin is an economical alternative to our highly purified tubulin (Cat. # TL238) 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 BK004 or BK011P or CytoDYNAMIX Screen™ BK004.
Purity
Protein 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 HTS02 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 Activity
An ASSAY UNIT is defined as the amount of HTS02 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 HTS02 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 HTS02. 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
S. Nazir et al. 2013. Brine shrimp lethality assay ‘an effective prescreen’: Microwave-assisted synthesis, BSL toxicity and 3DQSAR studies-based designing, docking and antitumor evaluation of potent chalcones. Pharm. Biol. 51, 1091-1103.
T.R. Butler et al. 2011. Neurodegenerative effects of recombinant HIV-1 Tat(1-86) are associated with inhibition of microtubule formation and oxidative stress-related reductions in microtubule-associated protein-2(a,b). Neurochem. Res. 36, 819-828.
M.C. Tuma et al. 2010. Antitumor activity of IMC-038525, a novel oral tubulin polymerization inhibitor. Transl Oncol. 3, 318–325.