Dear Yeast Gurus,
I have a problem and I am seeking advice from somebody who has
experience in studying the transport process in yeast. I am trying to
measure the uptake of inorganic phosphate (Pi) by yeast cells. At
present I am using the protocol of Ueda & Oshima (MGG 136:255, 1975),
which has been used by several groups. The problem I am encountering is
that of high background. We are using 32P (orthophosphate) as the
tracer. According to the protocol after adding the label you remove
samples of yeast culture and filter them thru nitrocellulose membrane to
retain the yeast cells, which are washed with several mls of the cold
mineral media, the filters are then dried and then counted by placing
them in scintillation fluid. However, most of the Pi sticks to the
filter (I have tested this by just filtering the media spiked with
labeled Pi) I am getting a background ranging from 20-30% of the label.
This makes it very difficult to measure the label incorporated by yeast
cells. So far I have already tried three different types of filters
(AAWP, 0.8u millipore; the Whatman 934-AH and GF/B glass fiber filters)
with no success in reducing the background.
Any suggestions regarding the types of filters that could be used to
circumvent this problem, any modifications in the washing conditions
after filteration, or any alternate procedures like centrifugation to
recover the cells (there is very little of them, 1 ml of 0.1 OD) would
be grately appreciated.
A ton of thanks in advance.
You can send me an e.mail at muchhal at omni.cc.purdue.edu or post it here
in this group.
Lost & Confused !!!!!!! :-(