Product Uses Include
The 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.
The kit contains sufficient materials for 30-100 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:
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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 not polymerize onto existing microtubules (MTs). This ratio is roµghly 10 volumes of buffer to 1 volume of cell pellet. Larger volumes of buffer are fine and in this kit the ratio is targeted 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.
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