Triton extraction - (Aug/14/2006 )

Hi,
I want to do an extraction on some cells in order to look at molecules binding to the cytoskeleton.
I´ve found and tried the following recipe for triton extraction:
20 mM PIPES pH 6,9
2mM EGTA
2mM MgCl2
1 mM PMSF
0,5% triton X-100

I think this solution might be too harsh and remove the proteins of my interest from their binding sites on the cytoskeleton. I would like to try a 0,2 % triton solution. Do I need to adjust the concentrations of the other ingredients as well?

---

No, I don't think so.

---

for simultaneous fix/extract (or a washout) i follow the protocol of Arcangelletti et al 1997 J Struct Biol 119:35-58
similar to your buffer but a few extras - worth a try?
10% triton, but i just adjusted the time.....

---

Hi aussieuk,

I´m affraid that so much triton would just be too harsh on the molecule binding to the cytoskeleton that I want to study. I am looking at a single protein which seems to bind very weakly and transiently to the microtubules. 0,5% already seemed too harsh when I compared it with my controls (cells that I fixed without extracting). That´s why I would like to lower the triton concentration to 0,2 %

I had a look at that paper from Arcangeletti and it seemed interesting although I do not think his protocol is suited for my own experiment.

Thanks anyway for the info!

---