adavison at selkirk.sfu.ca
Mon Apr 6 18:45:36 EST 1992
kristoff at genbank.bio.net (David Kristofferson) writes:
>DRM21 at MB1.BIO.CAM.AC.UK (David Micklem) writes:
>> I am making protein extracts from fruit fly ovaries and need to
>>cause depolymerisation of the microtubules. Does anyone know whether this
>>is possible to do by changing the buffer conditions, eg by including GDP or
>>metal ion chelators? Id like to avoid using particularly stringent
>>conditions like high salt/detergent levels if possible.
>>Many thanks for your help.
>It's been a while since I worked in this field, but fairly "low"
>levels of Ca++ (i.e., uM or less) should do the trick (I hope that
>memory has not failed me yet. Adding GDP would help, but I'd bet that
>smaller amounts of Ca++ would be more effective. Since you are making
<more good stuff deleted>
I used to prepare microtubules from sheep brains, and the problem
wasn't so much to get them depolymerized as to get them to polymerize.
My recollection of the calcium effect was as David K describes.
Moreover, polymerization required addition of GTP, Mg, (I think) and
warming to 37oC. Cooling to 0oC depolymerized. This is standard stuff
in the literature for microtubule preparation, so check that I didn't
get it backward. Else write me direct, & I can look it up. The bottom
line is that after a short period of benign neglect, most MT are gone,
due to hydrolysis of GTP in the homogenate.
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