Your question is not clear to me. It depends on what is your purpose for the isolation. I know there is an acetylated tubulin antibody which you may use for IP. There is also a microtubule stabilizing buffer and microtubule solubilizing buffer for other purpose. That is about all I know.
Hi Nilsa, if your purpose is to collect the stable microtubule population you may use a protocol we published in [Geeraert C et al. Journal of Biological Chemistry. 2010;285(31):24184–94] and adapt it to your cells by monitoring labile microtubule disassembly. Let me know if you need more information or if your objective is different.
Christian
I apologize for the vague description Ru-Jeng Teng. My purpose is to look at acetylated microtubules from pig RPE cells in order to check if motor proteins like dynein bind more tightly to these microtubules in cells treated with U18 or A2E or tubacin.
hm, motor complexes can be isolated by help of taxol-stabilized MTs... tubacin is the negative control? i saw a nice paper about it (taxol and motor complexes), but i forgot every details. first you check each line by IF, surely MDCK has a high level AcTub, i think. moreover, AcTub level is changing during culture stage.
Hi Nilsa,
Some former colleagues specialize now in isolation of tubulins with specific PTM. You can check their work "A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype". PDF is in free access in this link http://www.biochemj.org/content/449/3/643
In this protocol acetylation is augmented through the used of deacetylase inhibitors. Another alternative, a bit more complicated, is to produce recombinant aTAT1 and induce acetelytation of my formed in vitro.
Good luck
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