On 8-15-97 10:19a, Fergus Doherty wrote:
>Has anyone experience in incorporation of 32P into yeast phospholipids? If
>so can they point me to some references or give me some idea of culture
>conditions (unlabelled P free?) and concentratios of P32 reqired?
People commonly use 3H or 14C for phospholipid labelling unless one tries
to differentiate C-type phospholipase and D-type phospholipase. You may
lookup "Phospholipid Biosysnthesis" section in Molecular Biology of S.
cerevisiae (ed. Pringle, Broach, ?) for your starting point.
FYI, when I label phospholipids with 3H inositol or palmitic acid, I add
1uCi 3H/mL culture for a period of time, which depends on your purpose of
labeling. If the purpose is to prep phospholipids with an equilibrated
state, you may add lower strength of radioacivity for overnight culture
after several hours of preincubation (w/o label). If it is kind of
pulse-chase exp, you may need to set up a time-course ranging from few
minutes to several hours. In the latter case, you may combined labeled
culture with one or two vol of unlabeled culture for easier handling.
Hope this helps.
Namjin Chung, graduate student
Program in Molecular Cancer Biology
Dept of Pharmacology & Cancer Biology
Duke University Medical Center
n.chung at duke.eduhttp://www.duke.edu/web/ceramide