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Microtubule Binding Protein Spin Down Assay

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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:

  1. Tubulin, >99% pure, (Cat nr TL238)
  2. Microtubule associated protein fraction (positive control) (Cat nr MAPF)
  3. Bovine serum albumin (BSA) protein (negative control)
  4. General tubulin buffer (Cat nr BST01)
  5. Tubulin glycerol buffer (Cat nr BST05)
  6. GTP (Cat nr BST06)
  7. Microtubule resuspension buffer
  8. Paclitaxel (Cat nr TXD01)
  9. DMSO
  10. Salt-extraction buffer
  11. Manual with detailed protocols and extensive troubleshooting guide

Product Uses Include

Example results

The microtubul binding spin-down assay was tested by combining MTs with the a microtubule associated protein fraction (Cat nr MAPF) or with BSA. As expected, MAPFs co-sediment with the microtubules while BSA does not (Fig. 1 in the Gallery, "Related Content" section on the right)

Equipment needed

  1. TCA solution 50% (w/v) for protein precipitation if necessary.
  2. Centrifugation set-up capable of 100,000 x g at 4°C and 24°C, 50 -200 µl volume capacity.
  3. SDS-PAGE system.
  4. 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).
  5. Gel scanner for densitometric determinations.

Examples of publications where this product was used

 


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