Protein extraction from bone marrow
mavigozler at yahooDOT.com
Thu Oct 17 14:17:57 EST 2002
Presumably you are not interested in the protein but the nucleic acid, since
Trizol denatures the protein?
Bone marrow is essentially blood progenitors and assorted stem cells free of
connective tissue. Single cell suspensions can be obtained if you prevent
clotting by either rapidly washing freshly harvested marrow in balanced
salts solution, or by including heparin in the draw/flush with subsequent
washing. Let bone spicules settle out. You might try an easily done
Ficoll-Paque (or some other 1.077 g/ml prep) separation if you want to
enrich particular cell fractions (mononuclear, granulocytic, thrombocytic,
erythrocytic) before cell extraction.
I suppose if Trizol is not "reliable" in your view (I have never used it),
then older methods may do for you: after cell lysis incubate in proteinase
K with added detergent (very brief since you don't have a lot of connective
tissue), followed by protein removal using phenol:chloroform:isoamyl alcohol
(then maybe a back-extraction with ether) of high ionic strength aqueous
phase, then precipitation of the DNA or RNA; ribonuclease A treatment could
be done if it's only the DNA you want.
<mdsgshl2 at man.ac.uk> wrote thoughtfully:
> Apart from Trizol does anyone out there know of a reliable method for
extracting proteins from frozen bone marrow samples?
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