filter for heme

Deitiker, Philip R via methods%40net.bio.net (by pdeitik from bcm.tmc.edu)
Tue Jan 27 18:50:37 EST 2009


Heme likes to interact with polymers and filters are composed of polymers. 
Although I was never successful at recoving DNA from heparanized cells, I was able to recover cells
from extracellular, 'lysed cell', heme by spinning them across a glycerol step that separated heme bound material from cells.
If I recall correctly one needs 8 to 10% glycerol  to create a barrier robust enough to keep aggregate
heme bound material afloat, this was with whole heparinized blood in RBC lysis buffer. One can carefully layer the glycerol under the suspended cells and centrifuge them. For the most part the cells recovered only had heme bound on or within the cells. The DNA yeild was low, the iron apparently gets
to the DNA first. Heparin can apparently strip the iron off of the heme group, its bad news for DNA obtained from frozen cells, cause they are almost always permeated enough to let the heparin. 

Since your cell suspension is probably less dense than blood you probably could get away with a lower percentage of glycerol and I would recommend removing the cells and spinning them over the step gradient in a fresh tube at least once more. 
  
One trivial observation, when heparin is present with heme, the RBC membranes precipitate and will layer at about 10% glycerol concentration in RBC lysis buffer and the membranes will be lightly coated with heme. Its possible an amalgamation of polymers (RBC RNA, protein, etc) heparin, heme and
unbound iron. This indicates that heme or iron outside of the hemoglobin molecule or in the presence of heparin is quite capable of crosslinking to other materials.
apomyoglobin of course binds heme so . . . . . . 



-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Jiang, Xiaoxu
Sent: Tuesday, January 27, 2009 5:00 PM
To: methods from net.bio.net
Subject: filter for heme

Dear all,

I am a PhD student. I am working on a isotope labeled heme uptake experiment by bacteria cells. Now I have a problem about the filter for heme: after I incubate my cells with heme in medium, I am trying to filter out the unbound heme and take the membrane filter to the gamma counter with the cells
on it. But I find there are always a lot of unbounded heme sticks to the membrane and generates a high background, which pretty much ruins the experiments. I have tried several different kinds of membrane filters and right now I am using celluloseacetat filter but the background is still quite high.

I want to ask that what kind of membrane filter has the least non-specific binding for heme? And also, what kind of washing buffer is good to wash the filter to get rid of the non specific binding heme.

Thank you!
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