Biology Reference
In-Depth Information
16. ConA chromatography is performed in batch mode to reduce
material loss and to control the time of contact between the
protein sample and the resin. After incubation with the differ-
ent buffers, the resin is separated from the buffer by low speed
centrifugation to avoid bound-protein loss.
17. A part of the ConA lectin conjugated to the agarose resin was
shown to leak during chromatographic steps. Consequently, a
pre-washing step is added to remove loosely bound ConA and
limit the leakage of this lectin in elution fractions.
18. Proteins are briefl y separated by SDS-PAGE to get three
samples in order to concentrate proteins and to increase the
effi ciency of the tryptic digestion.
19. A silver nitrate staining can be carried out to detect low abun-
dant proteins not revealed by colloidal blue staining.
20. A silver nitrate staining protocol compatible with mass spec-
trometry analyses need to be performed [
35
].
21. It is important to infer the subcellular localization of the pro-
teins identifi ed by subcellular proteomics. It gives information
about the quality of the protein extract and allows a more reli-
able interpretation of experimental results. Whatever the care
taken to perform the preparation of a fraction enriched in a
subcellular compartment, there are always contaminant pro-
teins originating from other cell compartments. A predicted
subcellular localization should be validated only if the results
of two predictions are consistent.
22. Using software dedicated to prediction of functional domains
rather than BLAST homology search is usually more reliable.
Indeed, there might be wrong annotations due to partial
homology to a previously annotated protein [
36
].
Ackowledgments
The authors are grateful to the Université Paul Sabatier-Toulouse
3, France, CNRS, and INRA for support. A grant for the experi-
ments and the postdoctoral position of TDDB were provided by
the French
Agence Nationale de la Recherche
(Grant ANR-08-
BLAN-0193-01). The authors wish to thank Drs Benoît Valot and
Michel Zivy at the Plateforme d'Analyse Protéomique de Paris
Sud-Ouest
(PAPPSO)
for fruitful collaboration. Thibaut Douché
is acknowledged for his contribution to proteomics developments
in the lab.
