Hello,

I´m trying to obtain ISO (inside-out) membrane vesicles from CHO cells using a French press. I already tried once - with no results - on 1g CHO cells, lysis buffer composed by 50 mM KCl and ~2 mM MgCl2 (then I added also 2 mM EDTA because I read on a 1970 paper that divalent cations hamper the formation of ISO vesicles). The French press operated at 2000 psi. Is there something that doesn´t look good in this part of the protocol? Maybe I should add something else to the lysis buffer or the strenght of the press wasn´t high (or low) enough?

Thank you for your support,

Rocco