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.