Kits

Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps the you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for tubulin kits are shown below.  More citations are available on individual product pages.



Question 1: What is the best way to test whether my protein interacts with tubulin?

Answer 1:  To examine if your protein of interest interacts with tubulin, we recommend using one of our tubulin polymerization assays (Cat. # BK004P, BK006P or BK011P) for a sensitive measure of how the protein affects one or more of the three phases of polymerization (nucleation, growth and steady state).  Additionally, we recommend confirming that there are direct interactions between tubulin and your protein of interest with our microtubule binding protein spin-down assay biochem kit (Cat. # BK029). If tubulin heterodimer subunit binding is suspected (i.e. polymerization is inhibited) then one can coat streptavidin 96-well plates with biotinylated tubulin (Cat. # T333P) to create a tubulin subunit capture surface.  Then the compound or protein can be incubated in the well and binding can be visualized by an HRP detection system using an antibody (or a tag can be conjugated to the test protein) against the compound or protein of interest.  Keep in mind that buffer composition is also important as it will need to be low salt (approx 50 mM) and contain MgCl2 (or other divalent cation) and GTP to maintain subunit integrity.  For an example of a compatible buffer, see our recipe for general actin buffer (Cat. # BST01-001).  Alternatively, the protein of interest could be tagged with GST (avoid reducing agents) and conjugated to the plate and then incubated with biotinylated tubulin and visualized with HRP-conjugated streptavidin.

Question 2: What equipment do I need for the microtubule binding assay?

Answer 2:  Equipment required for the assay are an ultracentrifuge capable of centrifuging 50-200 μl volumes at 100,000 x g at 4°C and 24°C is needed.  Examples of such centrifuges are the Beckman airfuge with ultraclear tubes (Beckman, cat. # 344718), the SW50 ultracentrifuge rotor with adapters for ultraclear tubes (Beckman, Cat. # 344718) and a Beckman tabletop ultracentrifuge with TLA-100 rotor. 

Reagents and components not supplied with the kit include:  Laemmli reducing-sample buffer (1x stock, 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.001% bromophenol blue and 5% v/v β-mercaptoethanol) and (5x stock, 300 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 0.005% bromophenol blue and 25% v/v β-mercaptoethanol), polyacrylamide gels (12% or 4-20% gradient gels), SDS-PAGE apparatus and buffers, reagents for the detection of the “test” protein (Coomassie, silver stain or antibody), 50% (v/v) glycerol in Milli-Q water for Cushion Buffer resuspension and TCA solution (50% w/v) in Milli-Q water for protein precipitation if necessary.

The kit contents include: tubulin protein (Cat. # TL238), MAP fraction (Cat. # MAPF), BSA protein, general tubulin buffer (Cat. # BST01), cushion buffer (Cat. # BST05-001), salt extraction buffer, GTP stock (Cat. # BST06), taxol stock (Cat. # TXD01) and anhydrous DMSO.

Question 3: What is a good negative control for the microtubule binding reaction?

Answer 3:  Actin filaments are a good negative control because they are also negatively charged and pellet like the microtubules under high speed centrifugation.  Confirming that your protein of interest does not bind to actin filaments, but does bind to microtubules is additional confirmation that the test protein is a microtubule-associated protein (MAP).

For more information, click on the Documents tab above, or for Technical Support email tservice@cytoskeleton.com.