Am 27.01.2009, 18:59 Uhr, schrieb Jiang, Xiaoxu <jxx from ou.edu>:
> 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.
Is the heme bound to surface receptors of the bacteria or inside them? In
the latter case you can bring the background down by extensive washing
with a buffer containing "cold" heme. If the former, you have to be more
carefull, depending on the k_off washing may be possible, but I'd leave
out the cold heme. In any case you want a membrane with low binding for
heme (try polysufone), and you may want to preincubate the membrane with a
heme solution. I suspect however that most of the unbound heme you see is
in the fluid space between the cells, so washing is the only solution.
Some others have already suggested spinning the bacteria through a
gradient. I have done this with erys, using butyl phtalate (e.g. from
Fluka) as separation medium. Make a gradient of 120 ul 3 M KOH, 500 ul
butyl phtalate and add up to 200 ul sample. Spin 30 sec at maximum speed
(16,000 g) in an Eppendorf centrifuge. Remove the sample and most of the
butyl phtalate by aspiration (collecting the waste in a Wulff-bottle). You
measure the radioactivity in the KOH phase.
