Lysis conditions for cells?

Dear Experts,

What will I actually solubilize if I treat cells with a lysis buffer
that contains 1% tx100 as detergent?

More detailed: Grew hepatoma cells on dishes, washed them with PBS,
froze them in -80, scraped frozen layer into lysis buffer, pipetted up
and down a few times, flash froze in nitrogen, thawed in ultrasound
bath, spun down, I am using the supernatant and kept the pellet. Then
ran westerns with the supernatant.

This is a modified procedure where I used to treat adherent cells
directly with that buffer and the cytoskeleoton and the nuclei remained
on the dish.

I assume that I might get something off the membrane and then just have
the cytosol.

What will I get if I potter or ultraturrax the scraped off cells?

Thus, I am looking for some more detailed insights into detergent
fractionation of cells. Any insights you would like to share?

Best regards,

Wolfgang

In article <1153316958.483319.219040@b28g2000cwb.googlegroups .com>, "Wolfgang Schechinger" <[Only registered users see links. ]> wrote:


Ultrasound is unnecessary and probably even harmful because
it can lead to nuclear lysis.

With 1% Triton X-100 and moderate salt, you'll dissolve all cellular
membranes except nuclear. The pellet will contain nuclei and some
mitochondrial fraction. Cytoskeleton will be partitioned between
pellet and sup about (douncing and playing with salt conditions
can significantly affect this cytoskeleton distribution; salt above
~ 300 mM will start lysing nuclear membranes). Some
cholesterol-rich lipid aggregates do not dissolve and float to the
top during centrifugation.

If you are interested in cytosol only (e.g., only soluble proteins)
you will be better off doing freeze-thaw in isotonic buffer without
detergent and high-speed collecting sup.

HTH,

DK

Many thanks, Dima!

do you have a reference in your mind which is worth reading?

Wo

In article <1153347868.545898.322880@s13g2000cwa.googlegroups .com>, "Wolfgang Schechinger" <[Only registered users see links. ]> wrote:

No, with TX-100 it is all just "common wisdom" and long-term
personal experience. I do have a reference for freeze-thaw
permeabilization 'cause I wrote it :-) (Although the aim there
was to make cell ghosts that are physiologically intact but devoid
of cytosol so that exogenous factors can be added and assayed
for activity; still, I am quite sure that's the cleanest way of getting
"pure" cytosol).

If you need more input, just tell exactly what you are trying to do
and for what reason. Then it would be easier to guess what's
right and what's not.

DK