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radiolabeling proteins

Robert A. Bloodgood rab4m at virginia.edu
Mon Jul 23 15:12:40 EST 2001


Hi Mike,
    Regardless of the isotope, there are few things that one can get
INTO Chlamy in any appreciable amount.  Sulfate, acetate, arginine
are among them.  I have found 35S-sulfate a very cost effective way
to label Chlamy proteins to relatively good specific activity (if
cells are starved for sulfate in 90% sulfate-reduced medium first).
Pete Lefebvre pioneered this procedure; can be used as long term
labeling or as a pulse. 35S has advantages over 3H or 14C in terms of
autoradiography. However, I have done double labeling of Chlamy
proteins using long term (24 hr) 3H-acetate coupled with a pulse (1
hr) of 35S-sulfate (sulfuric acid).  Had to use fluorography to
detect the 3H on gels. [You can, of course, use 14C-acetate instead
of the 3H-acetate] Procedures are in my paper: Bloodgood, R.A., 1984,
Preferential turnover of membrane proteins in the intact
Chlamydomonas flagellum. Exp. Cell Res. 150: 488-493. I have labeled
Chlamy with radioactive arginine but it is EXPENSIVE and results in a
relatively low level of protein labeling.
Bob Bloodgood

On 16 Jul 2001 14:03:28 +0100 "Adams, Mike (Biology)"
<ADAMS at easternct.edu> wrote:

>  I fear this is going to expose my ignorance, but.. is there any reason why
>  people don't use 14C to label proteins in Chlamy?  I always understood that
>  this minimized labeling on non-protein compounds, but if the material is
>  going to be purified and run on gels, does it matter if other cell
>  constituents also get labeled?
>
>
>  Mike Adams
>  Biology Dept
>  ECSU, Willimantic CT 06266
>
>  If a stone falls on an egg, alas for the egg
>  If an egg falls on a stone, alas for the egg
>
>  ---



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