Dr. Tilo Brunnee (tilo at brunnee.IN-Berlin.DE) wrote:
: I attempt to isolate human mast cell heparin proteoglycan from human lungs.
: So far I enzymatically and mechanically prepare a single cell suspension
: from human lung, add 4 M Guanidine HCl and sonicate to disrupt the cells
: and dialyze against 1M NaCl/10mM Phosphate, pH 6.0 and apply this soup on a
: Dowex 1x2-200 ion exchange column equilibrated with the same buffer, elute
: with 3M NaCl/10mM Phosphate, dialyze against H2O and speedvac to dryness.
: I do have some questions regarding heparin:
: - Are there more efficient/more gentle ways to prepare highly sulfated
: - Are ther any contaminants (that bind to strong ion exchange at pH 6 in
: the presence of 1 M NaCl?) to expect from crude human lung?
: - Is it true that heparin side chains are bound to a protein core in human
: mast cells?
: - If so, is the heprain sidechain cleaved from the protein core during or
: before degranulation?
: - Is there any knowledge about the average lengh of the heparin molecules
: (or molecular weight) in humans?
: - Is there another source of heparin than mast cells in humans?
: - I wonder how commercially available heparin is isolated. That material
: seem to consist only of sulfated carbohydrates of relatively homogeneous
: I would be very grateful for some enlightment. Thank you!
If I wre you I would find a lot of references by Prof HRP Miller
(University of Edinburgh). His group deals a lot with Heparin, Mast
cells, and the associated proteins. There is also a prof Allen at
Newcastle who has techniques from dealing with GAGS, proeoglycans etc.
Medline searches should get you tthe appropriate references.
srps at galactose.mc.duke.edu
"Insert your own pithy phrase just about here!"