Biology Reference
In-Depth Information
software (Thermo Fischer Scientifi c), Spectrum Mill Proteomics
Workbench (SMPW) (Agilent Technologies).
11. All plant organs including roots, stems, leaves, fl owers, and
embryos can be used as explants. The ability to form callus
depends upon the organs and their developmental stages.
12. The disinfection process of plant material, which will be used
as explant source for initiating in vitro culture, varies depend-
ing on the tissue and the nature of the explants. It is therefore
necessary to perform a disinfection process optimization by
varying not only the proportion of the disinfecting agent but
also the time necessary for disinfection, to ensure success in
the establishment of in vitro culture. 70 % ethanol improves the
disinfected process since this increases the contact between
the plant material and the disinfected agent. The disinfection
process also improves by adding 0.1 % Tween 20 to 7 %
calcium hypochlorite solution.
13. Sealing Petri dishes with Parafi lm
®
to avoid dehydration.
14. In the case of grapevine (
Vitis vinifera
L.) SCC due to the
extracellular accumulation of resveratrol and related stilbenes,
secondary metabolites which are produced upon elicitation, a
step prior to protein extraction is needed in order to remove
these metabolites from the extracellular medium. The extracel-
lular medium is treated with ethyl acetate to extract these
stilbenes.
15. Once extracellular medium is depleted in stilbenes, residual
polyphenol compounds are removed by incubation with PVPP.
In general, plant tissues and plant cell cultures contain phenolic
compounds which can modify proteins through an enzyme-
catalyzed oxidative reaction interfering with protein extrac-
tion. PVPP or polyvinylpyrrolidone (PVP) removes phenolic
compounds by adsorption.
16. In case the aim of the study is to preserve the enzymatic activity,
protein precipitation should be performed in native condi-
tions; thus TCA protein precipitation method is not appropriate
for such analysis. Ammonium sulfate precipitation is a com-
monly used method for protein purifi cation based on the alter-
ation of the protein solubility. In the presence of high salt
concentrations, the proteins tend to aggregate and precipitate
out of solution. However, differential precipitation of proteins
using ammonium sulfate presents some limitations that should
be kept in mind as several contaminants will remain in solution
(e.g., nucleic acids) and many proteins remain soluble at high
salt concentration. This is the reason why this method is not
recommended when total protein representation is desired
being particularly helpful for protein purifi cation based on the
specifi c enrichment or pre-fractionation for a particular protein
