Product Uses Include
- To study the effects of pharmaceutical compounds on the ratio of Tubulin to Microtubules in cells.
- To study the effects of mutated cell lines versus their parent cell line for the change in ratio of Tubulin to Microtubules.
- To study the effects of physical alterations of environment on the ratio of Tubulin to Microtubules in cells.
Introduction
The most reproducible and accurate method of determining the amount of microtubule content versus free-tubulin content in a cell population is to use western blot quantitation of microtubule and free-tubulin cellular fractions. The general approach is to homogenize cells in microtubule stabilization buffer, followed by centrifugation to separate the microtubules from free-tubulin pool. Then the fractions are separated by PAGE and tubulin is quantitated by western blot. The final result gives the most accurate method of determining the ratio of tubulin incorporated into the cytoskeleton versus the free-tubulin found in the cytosol. This kit contains all the reagents to perform this assay.
Kit contents
The kit contains sufficient materials for 30-100 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:
- Lysis and Microtubule stabilization buffer
- GTP (Cat. # BST06)
- ATP (Cat. # BSA04)
- Protease inhibitor cocktail (Cat. # PIC02)
- Microtubule enhancing control solution
- Microtubule depolymerization control solution
- Control Tubulin Standard (Cat. # TL238)
- Tubulin monoclonal antibody
- SDS sample buffer (5 x)
- DMSO
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- Centrifuge capable of temperature controlled operation at 100,000 x g with volumes of 100 µl to 2 ml depending on the cell lysis volume
- SDS-PAGE minigel system and western blotting transfer apparatus
