cell lysis - How do I decide what solution to use? (Apr/26/2002 )
I am a chemist doing a bit of cell prep and analysis. I am trying to determine what cell lysis solution to use. My first thought was to use a simple detergent or hypotonic solution to lyse the cells...however, a quick literature search suggests that often more complicated solutions are used. Could someone please give me a brief explanation for the following cell-lysis-solution ingredients? I need to decide which of these are necessary for my purposes. Thanks in advance.
* Some solutions contain 150 mM saline. Why worry about osmolarity if you plan to kill the cells?
* Some solutions are buffered. Why worry about pH (with phosphate or citrate or HEPES or whatever) if you plan to kill the cells?
* Some lysis solutions contain EDTA. Why use it/what are you chelating?
* When is appropriate/necessary to inhibit proteases? How does one decide between the various options (e.g. DFP vs PMSF vs AEBSF, etc.)?
* How does one select the appropriate detergent(s). Sometimes one sees very simple mixtures (e.g. 0.5% Triton X) but sometimes relatively complicated (SDS + NP-40 + Na deoxycholate)...which should we use?
Sorry for the long post. For a reply, one sentence per topic should do--you will receive the eternal gratitude of a confused chemist.
(PS. If interested, I am presently analyzing CATH.a neuron cells for catecholamine content...but regardless of which lysis solution I use now, I am interested in the above topics for general info for the future.)
I can give a few ansewers to these questions.
First: you kill the cells, but precautions are taken to protect what you want: proteins!
* adding EDTA: chelate Ca2+, which may interfere in reaction, or activate proteases, like calpain.
* buffering + osmolarity: prevent that cells has shear stresses, cleving proteins.
* protease inhibitors: usualy a mix should do (maybe protease complete mini of Boehringer might be useful).
* detergents: have different properties. Usually, NP-40 (or Igepal-630), only make pores in the cytoplams membrane, so you wont have contamination of nuclear proteins.
thats something to start with...
Thanks for the info! Do you think this is a fair assessment: if I am interested in small molecules (catecholamines, in this instance), rather than proteins, that I do not have to be careful with pH, osmolarity, protease inhibitors, cold temp, etc.? Perhaps for my simple application (at this point) a simple detergent solution should do?