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cell lysis


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#1 adog3050

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Posted 26 April 2002 - 10:28 AM

Hi all:

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.

Thanks,
Aaron


(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.)


#2 Simon

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Posted 27 April 2002 - 03:08 AM

HI,

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...


#3 adog3050

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Posted 27 April 2002 - 09:58 AM

Simon:

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?

thanks,
Aaron





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