In one of the experiments, I need to show that in my treated cells, there is increase in soluble tubulin and there are less polymerized microtubules. What is the good protocol to fractionate them. I read few papers where they mention about incubating cells in Microtubule-stabilizing buffer + 0.5% triton for few minutes and scrap cells. Spin down, supernatant is soluble fraction. Dissolve pellet that will be the polymerized microtubule fraction. Other protocol talk about adding lammelli buffer in sup and pellet after separation. None of them seems to be working. the only positive result I see that in my western I can see tubulin band only in polymerized fraction lane. Soluble lane no band at all. However commassie stain show that there is plenty of protein. I have played with antibody concentrations as well.
Any expert in microtubule biochemistry who can give me some idea.
thanks
Neeraj