lyophilizing leaf tissue
Jeremy Coate
I need to lyophilize leaf tissue prior to shipping overseas. The tissue will
be used for analysis of lipids, which are vulnerable to endogenous lipases,
so I am planning to collect the tissue into liquid N2, freeze dry and ship
on dry ice to try to ensure that these lipases don't have a chance to do
their thing. I have never used a lyophilizer and have concerns that I'm
using it incorrectly, and that the samples are thawing.
I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on top
lautys
Lyophilizers suppose to fully dry up water content of the sample, so
"thawing" shouldn't be the concern.
lautys
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Dr Engelbert Buxbaum
You should have somebody show you how to use the instrument correctly.
That only takes 2 min, rocket science it ain't. Lyophilizers consist of a
vacuum pump and a cold trap. You connect your deep frozen sample to it,
and the solvent (water in your case) sublimates. The vapor is condensed in
the cold trap, which has to be cleaned from time to time. All the heat
coming into your sample from the environment is used to increase the
sublimation rate, the heat of evaporation ensures that the sample stays
frozen until all solvent has been removed.
In your case however, it perhaps would be more appropriate to extract the
lipids and ship those. See
@article{Rad-81,
AUTHOR= {M.S. Radin},
TITLE= {Extraction of tissue lipids with a solvent of low
toxicity},
YEAR= {1981},
JOURNAL= {Meth. Enymol.},
PAGES= {5-7},
VOLUME= {72},
LANGUAGE= {engl}
}
which uses cyclohexane rather than chloroform as in
@article{Bli-59,
AUTHOR= {E.G. Bligh and W.J. Dyer},
TITLE= {A rapid method for total lipid extraction and
purification},
YEAR= {1959},
JOURNAL= {Canadian J. Biochem. Physiol.},
PAGES= {911-917},
VOLUME= {37},
NUMBER= {8},
LANGUAGE= {engl}
}
The trick with all these procedure is to work under nitrogen, to prevent
lipid peroxidation.
Yoram Gerchman
While I totaly agree that extracting and sending the extract might be easier,
"Total" lipid extraction could be trickier then one would think. You might
watnt to take a look at this link
<http://www.lipidlibrary.co.uk/topics/extract/index.htm> and at the original
Folck papaer <http://www.jbc.org/content/226/1/497.full.pdf+html>.
Yoram
Quoting methods...@oat.bio.indiana.edu:
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> Date: Thu, 26 Nov 2009 12:34:24 -0400
> From: "Dr Engelbert Buxbaum" <engelber...@hotmail.com>
> Subject: Re: lyophilizing leaf tissue
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Dr Engelbert Buxbaum
<gerc...@research.haifa.ac.il>:
>
> While I totaly agree that extracting and sending the extract might be
> easier,
> "Total" lipid extraction could be trickier then one would think. You
> might
> watnt to take a look at this link
> <http://www.lipidlibrary.co.uk/topics/extract/index.htm> and at the
> original
> Folck papaer <http://www.jbc.org/content/226/1/497.full.pdf+html>.
> Yoram
Which only proves again that you should never cite a paper, or use its
method, without having it read, marked and inwardly digested ;-). Only
that way can you fully understand what you are doing.
Note however that the editorial confirms my initial hunch: the risk of
lipid peroxidation or hydrolysis is probably higher if frozen or
lyophilized tissue is shipped, compared to properly prepared and sealed
extracts. In addition of course, the methodological problems with
extraction are identical, regardless of whether that is done in the
sending or receiving lab.
The use of supercritical solvent (presumably CO2) mentioned in the
editorial is an interesting alternative, but beyond the reach of ordinary
laboratories. Thus the Radin method suggested by me initially, or the use
of ethanol/ethylacetate as mentioned in the editorial, are probably the
best options for the OP.