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IntroductionThe most reproducible and accurate method of determining the amount of microtubule content versus free-tubulin content in a cell population is to use western blot quantitation of microtubule and free-tubulin cellular fractions. The general approach is to homogenize cells in microtubule stabilization buffer, followed by centrifugation to separate the microtubules from free-tubulin pool. Then the fractions are separated by PAGE and tubulin is quantitated by western blot. The final result gives the most accurate method of determining the ratio of tubulin incorporated into the cytoskeleton versus the free-tubulin found in the cytosol. This kit contains all the reagents to perform this assay.
Kit contentsThe kit contains sufficient materials for 30-100 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:
Equipment needed
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
Fan et al., 2012. A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens. FEBS J. v 279, pp 2892-2904.
Liu et al., 2011. A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions. Mol. Biol. Cell. v 22, pp 2509-2519.
Mourad et al., 2011. Metabolic amplification of insulin secretion by glucose is independent of b-cell microtubules. Am. J. Physiol. Cell Physiol. v 300, pp C697–C706.
Roth et al., 2007. A microtubule-facilitated nuclear import pathway for cancer regulatory proteins. Traffic. v 8, pp 673–686.
Davis et al., 2005. Concurrent opposite effects of trichostatin A, an inhibitor of histone deacetylases, on expression of α-MHC and cardiac tubulins: implication for gain in cardiac muscle contractility. Am. J. Physiol. v 288, pp H1477-1490.
Question 1: When lysing the cells, how do I prevent existing tubulin monomers from polymerizing onto existing microtubules?
Answer 1: The microtubules/tubulin in vivo assay requires a constant cells-to-buffer volume ratio. Essentially the lysis step has to dilute the cellular extract so that the free tubulin does notpolymerize onto existing microtubules (MTs). This ratio is roµghly 10 volumes of bufferto 1 volume of cell pellet. Larger volumes of buffer are fine and in this kit the ratio istargeted at 50 volumes of buffer per volume of cells. Additionally, the average cell size is important in designing the experiment, so be sure to estimate the average cell size of your culture so that you can use it to calculate a good estimate for the volume of lysis buffer required.
Question 2: Is it possible to quantify the absolute amount of total tubulin in each of the experimental samples?
Answer 2: Absolute quantitation of cellular tubulin can be performed using the tubulin standard as a positive control at 50, 20, 10, 5 and 2 ng per lane.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
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