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a PD-10 desalting column equilibrated with 50 mM Tris/HCl
pH 8.0.
7. Depending on the biological system investigated, appropriate
combinations of Trx and NTR are selected. Preparations of
Trx and NTR should display electrophoretic homogeneity and
enzymatic activity should be confirmed spectrophotometrically
using Ellmans reagent as the final electron acceptor [ 24 ]. For
reduction of target disulfide bonds in barley seed tissue recom-
binant His-tagged barley HvTrxh1 and HvNTR2 produced in
E. coli were used [ 25 , 26 ].
8. A detailed protocol for preparation of StageTips is available
[ 27 ].
9. ICAT H /ICAT L ratios for barley thioredoxin targets were cal-
culated using the software MSQuant ( http://www.msquant.
sourceforge.net ) . Depending on the MS platform used a num-
ber of software tools are available. In our hands ICAT H /ICAT L
ratios between 0.86 and 2.64 were obtained for 199 labeled
tryptic peptides from Trx-treated barley embryo extracts and
cysteines in 104 peptides with ICAT H /ICAT L >1.22 were clas-
sified as Trx targets [ 3 ].
Acknowledgments
Karina Jansen and Mette Therese Christensen are acknowledged
for technical assistance. The Danish Research Council for
Technology and Production Sciences, the Carlsberg Foundation,
and the Centre for Advanced Food Studies (LMC) are acknowl-
edged for financial support.
References
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