microtubules / actin, centrifuging - (Oct/11/2006 )
Hello everybody!
I'm wondering, is it possible to spin down microtubules or actin polymers when centrifuged at 13000 rpm for 15 minutes? (1,5 ml ep, filled with 1 ml lysate)
And if it is, at which speed should I spin down my lysates if I want to loose the cell debris and keep the microtubular and actin structures?
Thanks in advance!
the answer to your first question is no. i don't remember the centrifugation conditions that i used for microtubules (the paper work is in another lab at this time, i could get it and be more specific but that shouldn't be necessary) but to pellet f-actin i would centrifuge at 140000xg for 1 hour (repolymerized from purified g-actin). 13000xg for 15 minutes just wouldn't cut it. for small volumes i would centrifuge in an airfuge or a beckman tl-100.
The problem is that I want to do a co-ip of a protein that is probably bound to microtubules and probably also to actin polymers. When I spin down at 13000 rpm (microcentrifuge) then most of my protein of interest is in the pellet. When I spin down for 2 minutes at 4000 rpm then most of the protein is in the supernatant.
The material I use is c.elegans embryos. They are hard to lyse. I grind them in liquid nitrogen and then sonnicate. But maybe it's still not enough then. Any tips of tricks?
the answer to your first question is no. i don't remember the centrifugation conditions that i used for microtubules (the paper work is in another lab at this time, i could get it and be more specific but that shouldn't be necessary) but to pellet f-actin i would centrifuge at 140000xg for 1 hour (repolymerized from purified g-actin). 13000xg for 15 minutes just wouldn't cut it. for small volumes i would centrifuge in an airfuge or a beckman tl-100.
The problem is that I want to do a co-ip of a protein that is probably bound to microtubules and probably also to actin polymers. When I spin down at 13000 rpm (microcentrifuge) then most of my protein of interest is in the pellet. When I spin down for 2 minutes at 4000 rpm then most of the protein is in the supernatant.
The material I use is c.elegans embryos. They are hard to lyse. I grind them in liquid nitrogen and then sonnicate. But maybe it's still not enough then. Any tips of tricks?
I wonder if it is useful to study Co-IP with polymers of F-actin or microtubuli as too much binds directly or indirectly to these scaffolds; ever tried immunocytochemistry to look for colocalization to actin or microtubuli?
the answer to your first question is no. i don't remember the centrifugation conditions that i used for microtubules (the paper work is in another lab at this time, i could get it and be more specific but that shouldn't be necessary) but to pellet f-actin i would centrifuge at 140000xg for 1 hour (repolymerized from purified g-actin). 13000xg for 15 minutes just wouldn't cut it. for small volumes i would centrifuge in an airfuge or a beckman tl-100.
The problem is that I want to do a co-ip of a protein that is probably bound to microtubules and probably also to actin polymers. When I spin down at 13000 rpm (microcentrifuge) then most of my protein of interest is in the pellet. When I spin down for 2 minutes at 4000 rpm then most of the protein is in the supernatant.
The material I use is c.elegans embryos. They are hard to lyse. I grind them in liquid nitrogen and then sonnicate. But maybe it's still not enough then. Any tips of tricks?
I wonder if it is useful to study Co-IP with polymers of F-actin or microtubuli as too much binds directly or indirectly to these scaffolds; ever tried immunocytochemistry to look for colocalization to actin or microtubuli?
I'm not looking for an interaction with actin of microtubuli, but my protein is probably bound to this....
Recently, i do study on actin, cell is lysed by lysis buffer 15min at 4 celsius centigrade, after that it is centrifuged at 12000rpm 10min, lots of actin is in supernant. i have read a article about actin, about half of actin is in supernant, and rest is in prepicitate. hope good luck with you!