Cat. # HTS02

Economical alternative to TL238 in primary screens for tubulin ligand drugs
High-throughput tubulin polymerization or depolymerization screens
Production of microtubule substrates for HTS motor assays

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 BK011 or CytoDYNAMIX Screen™ CDS01.

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.