Biology Reference
In-Depth Information
Leafs
Seeds
Pollen
Latex
Delipidation
Aqueous
Dilution
Detergent
±urea
Extraction
Ammonium sulfate
TCA
Acidic precipitation
Acetone
Protein
precipitation
Options
Desalting
Centrifugation
Fig.
1
Scheme of plant sample treatment as a function of extract origin prior to
protein recovery for further treatments (fractionation depletion or enrichment)
thus preventing proper protein capture, as well as in pigments,
lipids, polyphenols, and secondary metabolites that may interfere
with protein separation and analyses [
38
].
A large number of plant proteins are heavily glycosylated, thus
adding consequent problems of abnormal migrations in 2D elec-
trophoresis and diffi cult ionization in mass spectrometry. Various
cleanup protocols specifi c to plant extracts are available; however,
most protocols are not compatible with CPLLs. Figure
1
illustrates
possible options for four typical plant extracts used in conjunction
with CPLL. The following general rules should be adopted: (a) the
aqueous extraction should be performed in relatively low ionic
strength to prevent the solubilization of nucleic acids; (b) with
highly viscous material, such as latex and honey, a dilution is rec-
ommended; (c) when dealing with proteins that are engaged
within the cell wall, such as pollen proteins, some amounts of non-
ionic detergent (less than 0.5-1 %) and urea (less than 3 M) should
be used at a concentration compatible with CPLL; (d) a treatment
with phenol, associated with some amounts of polyvinylpyrrol-
idone, eliminates in general pigments and polyphenols.
Few examples are given in the literature with detailed technical
information [
29
]. A preliminary lipid removal step is particularly
recommended with plant seeds such as soya beans, peanuts, corn,
sunfl ower, and many others. Once undesired material is eliminated
plant proteins can be precipitated with ammonium sulfate or tri-
chloroacetic acid or acetone, or polyethylene glycol to collect
precipitates that are free or almost free of CPLL interfering sub-
stances. Acidic precipitation can also be operated just by acidifying
the solution with acetic acid at pH 3-4. It is here to be noticed that
the use of trichloroacetic acid as precipitating agent may induce
denaturation and therefore the loss of biological properties.
