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Product Uses Include
The contents of this kit will allow you to reproducibly prepare fluorescent microtubules of a predetermined mean length and of a predetermined fluorescent intensity. After polymerization the microtubules can be used directly or stabilized with taxol before use, depending upon the needs of your experimental system. This kit is ideal for the preparation of taxol stabilized fluorescent microtubules for use in an in vitro motility assay.
Figure 1. Rhodamine labeled microtubules created with kit BK007R
The kit contains enough materials for 50-200 assays depending on experimental setup. The following components are included:
Equipment needed Fluorescence microscope equipped with filters for rhodamine detection
Tong, T., Ji, J., Jin, S., Li, X., Fan, W., Song, Y., Wang, M., Liu, Z., Wu, M. and Zhan, Q. (2005). Gadd45a expression induces Bim dissociation from the cytoskeleton and translocation to mitochondria. Mol. Cell Biol. 25, 4488-4500.
Question 1: Do I have to stabilize the microtubules I’ve made with taxol?
Answer 1: We strongly recommend using taxol to stabilize the microtubules created with the fluorescent microtubules biochem kit (Cat. # BK007R). Microtubules are stable at room temperature when they are in the presence of a 10-20 mM taxol solution. In this state, the microtubules are stable for at least 24 h at room temperature (20-24°C). In the absence of taxol, microtubules are very sensitive to temperature variations. For example, microtubules stored at 4°C in the absence of taxol will depolymerize into tubulin heterodimers very rapidly. Thus, if you are performing experiments with non-taxol stabilized microtubules you should keep the temperature at 37°C.
Question 2: Can I alter the length of microtubules produced using the fluorescent microtubules Biochem Kit?
Answer 2: Yes, microtubules that are longer or shorter in average length than those resulting from the standard polymerization conditions described in the kit manual (Cat. # BK007R) can easily be produced. Below is a table that gives the polymerization conditions that are required to create microtubules of a given average length. Components of the reaction should be added in the order that they appear from left to right. It should be noted that all tubulin stock solutions that you are working with (including those that you create of a given stoichiometry) are at an initial protein concentration of 10 mg/ml
Table: Selected Conditions For Microtubule Polymerization
Average Microtubule Length Required (mm)
10 mg/ml Tubulin Stock (ml)
Microtubule Cushion Buffer
at 35°C (min)
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