Product Uses Include
- To determine whether a protein or compound binds to microtubules.
- To test various deletion mutants for their ability to still bind microtubules, then calculate their affinity for microtubules, and hence identify the microtubule binding site.
- In the presence of a non-hydrolyzable analog of ATP (kinesins) or GTP (dynamin) extract these from cells or show their binding affinity when mutated.
Introduction
This assay allows the identification of proteins that will bind to microtubules (MTs) in vitro. The assay relies on the fact that MTs will pellet when centrifuged at 100,000 x g. Therefore, any protein that is associated with the MTs will pellet with them during centrifugation. A simple SDS-PAGE analysis of the supernatant versus pellet fraction will identify if a protein is able to associate with MTs. The assay description given in this manual is for recombinantly expressed “test” proteins, however, the assay can be adapted for cell lysates or in vitro translation products.
It should be noted that in vivo confirmation of MT association should be obtained in order to confirm that the protein can be classified as a MAP. This association need not occur throughout the whole cell cycle and may even be developmentally regulated, indeed transient association of MAPs with microtubules is the norm rather than the exception.
Kit contents
The kit contains sufficient materials for 30-100 assays depending on assay volume. The following reagents are included:
- Tubulin, >99% pure, (Cat. # TL238)
- Microtubule associated protein fraction (positive control) (Cat. # MAPF)
- Bovine serum albumin (BSA) protein (negative control)
- General tubulin buffer (Cat. # BST01)
- Tubulin glycerol buffer (Cat. # BST05)
- GTP (Cat. # BST06)
- Microtubule resuspension buffer
- Paclitaxel (Cat. # TXD01)
- DMSO
- Salt-extraction buffer
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- TCA solution 50% (w/v) for protein precipitation if necessary.
- Centrifugation set-up capable of 100,000 x g at 4°C and 24°C, 50 -200 µl volume capacity.
- SDS-PAGE system.
- Detection system for protein of interest (coomassie is good for purified proteins, Western blot or silver stain for less pure or low abundance test proteins).
- Gel scanner for densitometric determinations.
