Thesis-PDF - IAP/TU Wien

Cell Treatment - Demembranation
In order to stop enzymatic activity and dissolve the membranes around the internal
cell parts the solution is mixed with a demembranation solution prepared as follows
(also see [115]):
The detergent Triton X-100 is added to a HEPES 1 -buffered solution (100mM
HEPES-KOH, pH 7.00; 20mM piperazine-N, N’-bis(2-ethanesulfonic acid); 10mM
EDTA; 50 mM sucrose; 1 mM dithiothreitol; 7,5% v/v glycerol) to give a final
detergent concentration of 4% v/v. It is then filtered and added to the cells (4:1),
which were previously suspended in 100 mM HEPES buffer (pH 7.00). Cells are
then washed repeatedly with the isolation solution to remove extracted chlorophyll.
This cycle of washing and reimmersion can be repeated during a couple of days to
weeks for better dissolution. Crystalline structures are not be dissolved.
Cell Explosion
Once the cells are washed free of dissolved membrane tissue, the cells must be
broken open. This can be achieved using the method of nitrogen pressurization
and rapid depressurization. The cells are stored in a sealed Ashcroft Duralife
pressure homogenizer (see figure 5.2) and then nitrogen is let flow in. During
pressurization nitrogen diffuses into the cell at an external pressure of 200 bar.
During rapid depressurization the cells burst (literally explode) because of their
internal pressure excess.
Figure 5.2: To the right the container for nitrogen pressurization of the
algae can be seen (also called a "Nitrogen-bomb"). The top piece as seen in
the left part of the image accommodates a pressure gauge and two valves to
pressurize the container and to rapidly depressurize it.
1 HEPES is an organic chemical buffering agent that is widely used in cell culture to maintain
physiological pH.
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