Microtubule / Tubulin In Vivo Assay Kit

AT

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.

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 roµ, ghly 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.

550

Centrifuge capable of temperature controlled operation at 100, 000 x g with volumes of 100 ul to 2 ml depending on the cell lysis volume SDS-PAGE minigel system and western blotting transfer apparatus