aj at biolsci.dundee.ac.uk (ANDY JOHNSTON) writes:
>Is there anyone on this list who could advise me as to the best method of
>making some CABP. I have read the lit fairly well and still cannot find a
>paper that describes the nuts and bolts of the method. Like how much, for how
>long.... the whole bit.
============
The classic paper on the synthesis of CaBP is:
Pierce J, NE Tolbert, R Barker (1980) Biochemistry 19: 934-942.
Virtually everyone uses this method verbatim. Variations occur with the
column matrix for purifying CaBP from CrBP, but few stray far from the
published procedure because leaning on it allows you to definitively say
which peak is CaBP. In many cases, separating CaBP from CrBP is
unnecessary. In this case, doing a classical Barium ppt. reaction using 3
to 3.5 equivalents of BaOAc (pH neutral with BaOH), maybe with 50% ethanol
to help ppt., is sufficient for purifying (the Ba is removed by the use of
a Dowex (50?) resin to Ba for H+.
Ultimately, you must make a few educated "guesses" at various stages of the
protocol -- especially when using 14C-CN to make 14C-CaBP. Your best bet
is to take some RuBP and try the protocol as published using your "best
guess" at just what to do at various stages.
About the only critical step is ensuring you've made the lactone. This
takes solid procedures to ensure that all excess water has been removed,
then maintenance of a low pH to ensure that the lactone doesn't open up
while being eluted from the column.
>If anyone has any thoughts on the use of CABP as a method of determining total
>RUBISCO levels I would be grateful.... or any other methods on this subject.
The use of CaBP to quantify Rubisco levels is well-established -- we do it
in our laboratory routinely.
--
Eric Larson | University of Illinois at Urbana-Champaign
USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419
Fidonet: 1:233/4.1 | My opinions are my own, but correct :-)
