32Pincorporation into yeast phospholipids

[Namjin Chung]

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?
>
>Thanks
>
>-- 
>Fergus Doherty,
>Dept Biochemistry,
>Nottingham University,
>
Dear Fergus,

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.edu
 http://www.duke.edu/web/ceramide
_______________________________________