cell lysis - 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.)
Cell Lysis Buffers depend on what you want to do with your with your lysate:
The EDTA is usually to chelate Ca ions involved in intercellular and intra-cellular adhesion so you get better break down of cells. May also chelate ions that are co-factors of certain enzymes.
Protease inhibitors are used to prevent the protein being degraded if it is a protein that is of interest to you. The different inhibitors used have different specifices for inhibiting certain families of proteases e.g. PMSF for serine proteases.
Proper pH control is needed to mantain activity of proteins for enzyme assays etc. so that they don't unfold and become inative or in the case of Westerns lose their antigenicity. Same for maintaining osmolarity.
Again with the choice of detergent you can seletively disrupt different membranes to release contents.