Many papers use complicated methods to isolate microtubule associated proteins (MAPs), such as column chromatography and suspension cell culture, etc.
Does anyone have a protocols that use easy and cheap methods to isolate MAPs from Arabidopsis leaf? I need to confirm the interaction of my MAP and its partner proteins by co immunoprecipitation (CoIP). Furthermore, use protoplast-CoIP-LC-MS/MS to find more interacting proteins of my MAP.
however, I can not detect my MAP-YFP by normal proteins extraction methods (buffer: Tris-HCl, NaCl, MgCl2, NP-40, PMSF and protease inhibitor. centrifuge at 13000 rpm). only use extraction buffer with urea can detected by Wastern.
is possible that my MAP sediment with pellet when centrifugeation?
Hi Yu Yuan,
you definitely need to use the buffers described in the literature you refer to, since these allow to conserve and isolate microtubules in the first place. After that you may have several choices, depending on what tools you have. You mention CoIP, i.e. you probably have antibodies against your protein and that can test whether you find tubulins in a subsequent Western blot or vice versa.
Since you mention also protoplast expression, you can also tag your protein and try e.g. colocalization of fluoresence if you use e.g. a FP-MAP line as a source for your protoplasts.
Finally, if you use a buffer that conserve MT (maybe even stabilize them with drugs), then you should be able to precipitate them in an ultracentrifuge. There are protocols in the literature.
Best wishes and good luck,
Tony Schaeffner
Muenchen, Germany
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