Dear All,Back to the Top
I am looking for a method for preparing intestinal microsomes. I don't
know how to separate the mucosa from the whole intestinal sample.
Does anyone have a good advice ?
Check this reference:Back to the Top
Lu X, Li C, Fleisher D. Cimetidine sulfoxidation in small intestinal
microsomes.Drug Metab Dispos. 1998 Sep;26(9):940-2.
I hope this helps.
Kishore K. Khan, Ph. D.
8609 Cross Park Drive
Austin, TX 78754
Tel: (512) 615 2332
Fax: (512) 834 7767
Nicolas,Back to the Top
My favourite method is to excise the small intestine and flush out the
contents with ice cold buffer (e.g. PBS). I then slit the tissue
lengthwise and place it, mucosal side up, on a chilled glass plate.
I then use the edge of a microscope slide to scrape away the mucosa
from the underlying connective tissue. Microsomal fractions can then
be prepared from this scraped material. Be sure to use protease
inhibitors (PMSF or AEBSF, at least) in the homogenisation buffer.
Adding DTT also improves things and appears to have some mucolytic
activity. Some people suggest adding heparin but I have not found
it to be useful.
A colleague of mine who has performed this on a regular basis, and
uses the microsomal fractions for western blotting, recommends
normalising results to the expression of villin. Unless you are very
consistent with your scraping procedure trying to normalise to
microsomal protein concentration is probably not that useful.
I hope that this helps.
All the very best,
Bernard Murray, Ph.D.
Senior Research Investigator
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
There is a good reference:Back to the Top
Zhang, Q. Y.,Dunbar, D.,Ostrowska, A.,Zeisloft, S.,Yang, J.,and
Kaminsky, L. S.
Characterization of human small intestinal cytochromes P-450, Drug
Metab Dispos, 1999, 27(7), 804-809.
Kenan Gu, Ph.D.
Phone (732) 661-2238
Tissue Transformation Technologies
Thanks a lot for your prompt response. I will try with your method.Back to the Top
Nethertheless, I'm afraid it will be difficult because I work with human
samples, quite small, and frozen at -80\0x221EC. I think it will be difficult
ensure that I have only the mucosa.
PharmD, PhD student
Laboratoire de Pharmacologie - DEXO
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (email@example.com)