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Product Uses Include
This kit is intended for those researchers who are not accustomed to working with tubulin protein in vitro. The contents of this kit will allow you to reproducibly prepare microtubules of a predetermined mean length. After polymerization the microtubules can be used directly or stabilized with taxol before use, depending upon the needs of your experimental system.
The kit contains enough materials for 8-200 assays depending on experimental setup. The following components are included:
van Horck, F. P., Ahmadian, M. R., Haeusler, L. C., Moolenaar, W. H. and Kranenburg, O. (2001). Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules. J. Biol. Chem. 276, 4948-4956.
Question 1: Can I make microtubules of a different length with the Microtubules/Tubulin Biochem Kit (Cat. # BK015)?
Answer 1: Yes, the Microtubules/Tubulin Biochem Kit (Cat. # BK015) can be used to produce microtubules of variable length. Below are instructions.
Preparation of Microtubules of a Given Length Distribution
1) Decide the mean length of the microtubule (MT) population you will require. Generally a mean length distribution of 5-10 μm is used for MT binding assays and 2 to 5 µm for MT motility assays (see Table below).
2) If you are going to stabilize the MTs with taxol, aliquot 90 μl of General Tubulin
Buffer (Cat. # BST01-010) into a clean tube (labeled TX buffer) and add 10 μl of 2 mM stock taxol. Mix well by pipetting and keep at 35-37°C.
Note: Each condition for MT polymerization described below requires one 250 μg tube of tubulin (supplied in kit, Cat. # TL238-A).
3) Prepare the appropriate MT Buffer formulation as described in the Table below for your choice of MT length. Keep on ice.
4) Briefly centrifuge (16,000 x g for 5 s) one 250 μg tube of tubulin (Cat. # TL238-A) to collect the product at the bottom of the tube.
5) Resuspend one tube of tubulin with the required volume of MT Buffer shown in the Table, column 2.
6) Incubate the tube on ice for 10 min to completely resuspend the protein.
7) Incubate the tube at 37°C for 40 min; this will give you a MT stock solution of the required length.
8) To stabilize the MTs, add 1/10th the volume of TX Buffer made in step 2 and incubate for a further 10 min at 37°C (optional).
9) The MTs can now be used directly in your assay of choice.
Note: Taxol stabilized MTs will be stable for several days (Store at room temperature, do not store at 4°C); however the length distribution will change over time (the MTs will become longer). If this is not a problem for your experiments, then these MTs can be used for 4 days. If however, you require MTs of a consistent mean length distribution, we recommend using the MTs within 6-7 h. Anti-microbial agents (50 mg/ml ampicillin and 5 mg/ml chloramphenicol) can be added if the MTs are going to be kept for longer than one day.
Table. Selected Conditions for Microtubule Polymerization
|MT Buffer Formulation|
Volume of MT Buffer to add to one tube of tubulin
Buffer to add
Tubulin Buffer to add
100 mM GTP to add
Question 2: After microtubules are prepared and stabilized with taxol using the Microtubules/Tubulin Biochem Kit (Cat. # BK015), can they be stored for later use?
Answer 2: Yes, taxol-stabilized MTs will be stable for several days. The taxol-stabilized MTs should be stored at room temperature. Do not store at 4°C as they will depolymerize. Be aware that the length distribution will change over time (the MTs will become longer). If this is not a problem for your experiments, then these MTs can be used for 4 days. If however, you require MTs of a consistent mean length distribution, we recommend using the MTs within 6-7 h. Anti-microbial agents (50 mg/ml ampicillin and 5 mg/ml chloramphenicol) can be added if the MTs are going to be kept for longer than one day.