Dae Kyun Ro wrote:
> Hi, protein netters. I want to rupture my cells (insect cells such as sf9,
> ld, hi-5) as gentle as possible. Osmotic lysis seems to be the way to go,
> but I don't know how I can design the lysis buffer. I looked up some papers
> about the osmotic shock, and I have found a variety of different lysis
> buffers. I don't want my cells suddenly ruptured. It would be ideal that
> the cells gradually take up water in certain period of time and broken
> open smoothly at some point. Gentle lysis is critical in my experiment
> because I want to analyze the multienzyme complex on ER. The nature of
> protein-protein interaction may be very weak.
>> Can anybody tell me good reference or idea? Thank you in advance.
>> **********************************
> Dae Kyun Ro
> 439, 6335 Thunderbird Cres.
> Vancouver, BC
> Canada V6T 2G9
Several years ago I used to rupture protoplasts isolated from plant stomatal
guard cells. The idea was to rupture them slowly to get only cytosolic
content out.....w/o serious plastids damage etc.
Each organism has different osmotic pressure, so..... I think first of all you
need to determine that using osmometer and then osmotic activity of the buffer
you want to use. Then you can....play with salt concentration to gradually
decrease it in buffer....and observe all the time your cells under microscope.
In such a way you...probably will be able to fix the salt concentration and
treatment time...when cells rupture slowly and gently. That's a matter of
test and trial, sure. This is in general, details I can ask to my former
students.
Good luck
L. Darjania
