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[Sorry for the break in message flow - poor email access while
traveling, I'm back in the office so I should be able to fix things
quicker now :-) hopefully not too many missed or duplicate messages -
db]
The following message was posted to: PharmPK
Dear group,
We would like to assay a compound concentration in kidney, liver,
brain and solid tumors after drug administration in nude mice. Could
anyone recommend some protocols or literature about methods of tissue
homogenization and precipitation of protein & lipids?
Thank you.
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The following message was posted to: PharmPK
We routinely use beadbeater to homogenize tissue samples up to 400 mg.
For more information, you can go to www.biospec.com. Hope this helps.
Yinuo Pang, Ph.D.
Associate Staff Scientist
Preclinical Research
ICOS Corporation
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The following message was posted to: PharmPK
To assay "compound concentration in kidney, liver, brain and solid
tumors",
please consider pitfalls of "tissue homogenization". All of these
tissues are
composed of a number of heterogeneous cells populations with different
functions. As Lloyd Roth stated:
"Don't homogenize the brain. The brain you are homogenizing may be
your own."
Cited from: Stumpf WE: Drug Localization in Tissues and Cells.
ISBN 0-9740515-0-0.
www.unc.edu/~stumpfwe (or: www.unc.edu/~stumpfwe/bio)
I discussed this in detail in this book.
Walter E. Stumpf, Chapel Hill
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The following message was posted to: PharmPK
Hi Lori,
Biospec.com sells several types of beadbeaters. The one in our lab
(Mini-BeadBeater-8) can hold up to 8 sample vials. Those vials are
disposable plastic vials with a capacity of 2 mL. Generally, tissue
samples (up to 400 mg each) are placed into the vials together with a
mixture of 2.4 mm and 1.0 mm beads (also disposable), then certain
amount of tissue homogenization solution (usually we use 20% MeOH in
H2O) is added into each vial. Several 60-sec pulses are then used to
homogenize the tissue. To minimize the heat generated by the movement of
the beads, you can put the samples in an ice/water bath before and after
each pulse. Following homogenization, you can transfer your homogenized
samples into centrifuge tubes to perform further extraction process.
Since both the vials and the beads are disposable, contamination or
instrument cleaning shouldn't be an issue. This has worked well for most
of our tissue samples. If you are concerned about the compound stability
due to enzyme liability from the tissue compartments or simply higher
temperature, I suggest you try the Bio-pulverizer (also available on
biospec.com) to crush the frozen tissues into powders and then add your
extraction solution which usually contains high proportion of organic
solvent (such as ACN) to deactivate the enzymes.
Hope this helps.
Yinuo Pang, Ph.D.
Associate Staff Scientist
Preclinical Research
ICOS Corporation
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)