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.
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