Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Lysis conditions for cells?

Lysis conditions for cells? - Protocols and Methods Forum

Lysis conditions for cells? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-19-2006, 01:49 PM
Wolfgang Schechinger
Guest
 
Posts: n/a
Default 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

Reply With Quote
  #2  
Old 07-19-2006, 02:28 PM
DK
Guest
 
Posts: n/a
Default Lysis conditions for cells?

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
Reply With Quote
  #3  
Old 07-19-2006, 10:24 PM
Wolfgang Schechinger
Guest
 
Posts: n/a
Default Lysis conditions for cells?

Many thanks, Dima!

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

Wo

Reply With Quote
  #4  
Old 07-20-2006, 06:13 AM
DK
Guest
 
Posts: n/a
Default Lysis conditions for cells?

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
Reply With Quote
Reply

Tags
cells , conditions , lysis


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Cell Lysis for Enzyme Activity oBWhat Cell Biology and Cell Culture 4 11-08-2012 01:40 PM
running ssDNA (30-1000nt) in denatured conditions TC Protocols and Methods Forum 1 11-19-2008 08:10 AM
Galileo (NOT Einstein) is inventor of Second postulate of Relativity AJAY SHARMA Physics Forum 0 11-05-2006 02:22 AM
alt.astronomy, alt.sci.physics, alt.sci.physics.new-theories, AJAY SHARMA Physics Forum 0 11-05-2006 02:20 AM
Reply to: Lysis conditions of the cells/ Wolfgang post RNA Master Protocols and Methods Forum 1 08-31-2006 09:40 AM


All times are GMT. The time now is 02:05 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13235 seconds with 16 queries