Is there a way to detach proteins from microtubules (spindle microtubules) from a nuclear extract in vitro? I've tried to do this in vivo by treating living cells with nocodazole before extraction, but this did not cause significant release of my protein of interest into the supernatant.
The catch here is that I have to do this without detergents.
Any advice about this detachment in vitro would be greatly appreciated. Thank you.
High salt or denaturants such as urea and guanidine hydrochloride might work to dissociate the proteins.
Other drugs besides nocodazole that depolymerize microtubules include colchicine and vinblastine.
Thanks for the information, Adam.
I've tried high pH (8.5 -10.2) to dissociate the proteins from microtubules in nuclear extracts, but I was unsuccessful thus far. High salt was nixed by my PI because that causes microtubules to aggregrate and we don't want that.
There's something I forgot to mention - we also need conditions that won't affect vesicles. We suspect that our protein of interest has a vesicle around it and we're interested in pulling that vesicle down and characterizing it. So if urea or G-HCl won't affect vesicular structures, they might work.
I seem to recall that the method for purifying tubulin involves polymerization and depolymerization cycles, accomplished by warming and cooling. Can you use that procedure to depolymerize microtubules, with the expectation that the associated proteins will dissociate from depolymerized tubulin?
I will look into using temperature cycles for purification, Adam. Thanks again for the information!
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