I want to run a ROS assay on tissue lysates from mice. However, the assay is sensitive to detergents and calls for "low" (~1 uM) concentrations of background organics.
Consequently, I do not want to use the common RIPA buffer/protease inhibitor cocktail due to detergents and 1-10's millimolar amounts of interfering organics. I have a couple of questions.
(1) Does anyone know of a method/protocol to dissociate/lyse cells from mouse tissue in PBS but with minimal detergents and organics (ex: protease inhibitor/EDTA/Chelators)?
(2) Are there alternatives to protease inhibitors? I want to keep all proteins intact for a BCA assay.
The tissue samples are collected, snap frozen and kept at -80 before use.
If you need a simple protocol to dettach and lyse animal cells without detergents, you must try the use of ET-buffer...It is a very simple buffer (10 mM Tris pH 8.0 with 1 mM EDTA)...You must use the EDTA to dettach the cells, just some lateral strokes at the culture bottle is all you need to dettach...The hypotonic lysis do the rest...After some shakes, all my Vero ( African Green Monkey Kidney cells) were promptly dettached and lysed... The protocol is easy : Remove the culture medium, wash the cells twice with calcium free PBS and change to 15-20 ml of ET-buffer...after that procedure the cells will be detached and hypotonic lysed after 10 minutes... It worked well for me with kidney and intestinal cells.
I recommend you reading the " Methods in enzymology" which dealt with the different methods that are used to lyse membrane from different species ( Plants, Bacterial and animals) As far as I remember it is volume 20 or 22 it has very important detailed information related to this subject.
Good Luck
Have you tried the traditional physical disruption methods to break open the cells? Introducing the Lysozyme may also be an option.
- Badges
- Science method