Important Notice:
This product is no longer available. Individual components listed below are available for purchase.
Product Uses Include
- Create fluorescent microtubules for in vitro motility assays.
- Use separate components for separate experiments (e.g. microinjection and biochemical assays).
- Teaching aid for undergraduate or graduate classes.
IntroductionThe 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
Kit contentsThe kit contains enough materials for 50-200 assays depending on experimental setup. The following components are included:
- 5 x 20 µg Rhodamine Labeled Tubulin (Cat. # TL590M).
- 2 x 250 µg Tubulin Plus Glycerol (Cat. # T240).
- General Tubulin Buffer (Cat. # BST01).
- GTP in PEM Buffer (Cat. # BST06).
- Tubulin Glycerol Buffer (Cat. # BST05).
- Taxol stock
- Anhydrous DMSO
- Antifade
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed Fluorescence microscope equipped with filters for rhodamine detection
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
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) | General Tubulin Buffer (ml) | Microtubule Cushion Buffer (ml) | Time of Incubation at 35°C (min)
|
2.0 | 2.0 | 0 | 2.0 | 10 |
6.5 | 2.0 | 0.6 | 1.4 | 20 |
16 | 2.0 | 1.7 | 0.3 | 30 |
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
