in vivo 32P labeling of Chlamy - methods

Robert A. Bloodgood rab4m at UVA.PCMAIL.VIRGINIA.EDU
Tue Jul 13 21:16:36 EST 1993


    I recently responded to a request from Ward Lutz in Jeff Salisbury's lab
for information on improving 32P labeling of Chlamydomonas and thought I
would share our current method with the Chlamy Newsgroup. We grow
Chlamydomonas reinhardtii, strain pf-18, in medium I of Sager and Granick,
what anyone ever associated with Joel Rosenbaum's lab calls M (minimal)
Medium.  For 32P-labeling we grow small cultures for 3 days in medium with
10% of the normal level of each of the two phosphate stocks plus 10 mM Hepes,
all adjusted to pH 7.0-7.2.  We then use these small cultures to start 8
liter cultures which are grown for 3 days, again in medium with 10% of the
normal level of cold phosphate plus 10 mM Hepes.  We don't see any obvious
reduction in the rate of cell growth with this phosphate depleted medium.  We
collect cells into M medium lacking all phosphate and containing 20 mM Hepes.
We add 5 mCi of NEX-053 (New England Nuclear) Orthophosphoric acid in water
to 200 mls of cells in medium minus phosphate plus Hepes at 4 X 10 to the 7th
cells/ml giving a final concentration of 25 microCuries/ml. For some
experiments, the cells will be divided into two separate 100 ml batches - we
do the phosphorylation (and any subsequent drug treatments) in 250 ml plastic
round bottom centrifuge bottles.  We predilute the 32P-orthophosphoric acid
stock with 5 mls of medium before adding it dropwise to the cells rotating on
a shaker.  We usually incubate cells with 32P for 60 minutes at RT on a
shaker in front of bright fluorescent lights, although we find a 10 minute
incubation gives plenty of incorporation into proteins and no difference in
the 2D pattern of flagellar phosphoproteins relative to 60 minutes.  At the
end of the 60 minute labeling time, cells are diluted with equal volume of
cold medium, centrifuged in the cold and resuspended in a small volume of a
ice cold Witman's HMDS (4% sucrose) medium and then deflagellated by addition
of dibucaine per procedures in Witman Methods in Enzymology article.
Immediately after deflagellation, we add an equal volume of an ice cold STOP
solution containing 20 mM Hepes, 20 mM EGTA, 200 mM NaF, 4% sucrose,
Aprotinin, Microcystin and Okadaic acid to slow down the kinases,
phosphatases and proteases.  We go on to purify and fractionate the flagella,
although your interest may well lie with the cell bodies.  We get plenty of
label into flagellar proteins, even though we are not regenerating the
flagella in the presence of the label.  Our 2-D gels of flagellar
membrane-matrix phosphoproteins are usually exposed to X-ray film with one
Cronex enhancing screen for less than 12 hours.  Spots on the 2D gels are
plenty hot to do phosphoamino acid analysis with exposure times of a week. I
am sure this procedure can be scaled down considerably but we often go on to
fractionate or immunoprecipitate the 32P-flagellar membrane-matrix fraction
so we want to start with lots of flagella.




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