Cat. # BK029

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.

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

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.
   
   

Example results
The microtubul binding spin-down assay was tested by combining MTs with the a microtubule associated protein fraction (Cat. # MAPF) or with BSA. As expected, MAPFs co-sediment with the microtubules while BSA does not (Fig. 1)

Figure 1. MAP Spin-Down Assay using BK029. Microtubules were mixed with buffer (MT), MAP proteins (MT + MAPF) or BSA (MT +BSA) and MTs were pelleted by centrifugation at 100,000 x g. Supernatant (S) and Pellet (P) fractions were examined by SDS-PAGE. MAP proteins, but not BSA, bind to MTs and co-precipitate. MAP (MAPF) or BSA (BSA) proteins alone do not pellet.

Examples of publications where this product was used
Cho, H. P., Liu, Y., Gomez, M., Dunlap, J., Tyers, M. and Wang, Y. (2005). The dual-specificity phosphatase CDC14B bundles and stabilizes microtubules. Mol. Cell. Biol. 25, 4541-4551.

Lansbergen, G., Komarova, Y., Modesti, M., Wyman, C., Hoogenraad, C. C., Goodson, H. V., Lemaitre, R. P., Drechsel, D. N., van Munster, E., Gadella, T. W., Jr. et al. (2004). Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J. Cell Biol. 166, 1003-1014.

Monzo, P., Gauthier, N. C., Keslair, F., Loubat, A., Field, C. M., Le Marchand-Brustel, Y. and Cormont, M. (2005). Clues to CD2-associated Protein Involvement in Cytokinesis. Mol. Biol. Cell 16, 2891-2902.

Pennetta, G., Hiesinger, P., Fabian-Fine, R., Meinertzhagen, I. and Bellen, H. (2002). Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron 35, 291-306.

Ziegelbauer, J., Shan, B., Yager, D., Larabell, C., Hoffmann, B. and Tjian, R. (2001). Transcription factor MIZ-1 is regulated via microtubule association. Mol. Cell 8, 339-349.