Microtubules (Taxol Stabilized and Lyophilized)

On Arrival: 4°C

Question 1: If the pre-formed microtubules are resuspended, aliquoted and snap-frozen as directed, will they depolymerize and need to be re-polymerized or is the polymerized state maintained during one freeze and thaw cycle?

Answer 1: The pre-formed microtubules ((Cat. #MT002) will not depolymerize upon one freeze thaw cycle. Thaw rapidly in a room temperature water bath and then keep at room temperature until ready to use. However, when aliquoting for snap-freezing, pipette smaller volumes (10-20 ul) since the concentration of the microtubules will be 1 mg/ml (based on datasheet instructions).

Question 2: What is the difference between the two different versions of pre-formed microtubules (Cat. #MT001 and MT002)?

Answer 2: The only difference between the MT001 pre-formed microtubules and the MT002 pre-formed microtubules is the source of the tubulin used to product the microtubules. MT001 uses bovine brain and MT002 uses porcine brain. In side-by-side comparisons (please see the MT002 datasheet), the microtubule-activated kinesn ATPase activity (Cat. #BK053) of MT001 (bovine) and MT002 (porcine) microtubules was compared. Both sources of microtubules produced the same results with this kinesin ATPase end-point assay (Cat. #BK053).

Substrate for activation of kinesin ATPases (see Fig. 1)
Determination of IC50s for kinesin inhibitors
Used in Cytoskeleton, Inc's kinesin ELIPA and End point ATPase assays (Cat. #BK060 & BK053)
Substrate for discovery and characterization of microtubule binding proteins (MAPs) (see Fig. 2)
Ideal for HTS applications

MTs serve as a substrate for kinesin motor proteins. Kinesin motor proteins orchestrate a wide range of kinetic events within a cell. They have been shown to move cargoes such as chromosomes and vesicles along MT tracks. Kinesins operate by utilizing the energy of ATP to hydrolysis, an activity that is greatly enhanced in the presence of MTs. An MT activated Enzyme Linked Inorganic Phosphate ATPase (ELIPA) assay (Cat. #BK060) can therefore be used as a test for the biological activity of MT001 (Fig. 1). The motor protein used in this assay is Kinesin Heavy Chain motor domain (KHC: Cat. # KR01). MT001 is also an excellent substrate for detecting microtubule binding proteins, see Fig. 2.

Figure 2. Microtubule (MT) binding protein spin-down assay using MT002. >80% of MT002 (arrow: Tubulin) is in pellet (P) after spin-down. MAPs (Cat. # MAPF) bind to MTs and end up in pellet while BSA does not and stays in supernatant (S).