Biology Reference
In-Depth Information
6. Sample-spotting solution: 50 % (v/v) acetonitrile, 0.1 % (v/v)
TFA.
7. Matrix solution:
-cyano-4-hydroxycinnamic acid (7 mg/mL
in sample-spotting solution).
α
3
Methods
The extraction of the proteins from the plant sample is a crucial step.
Many extraction methods exist for tree samples, and none can be
considered as “THE” protocol. It is thus very important to optimize
the extraction protocol and the sample handling to avoid interfering
substances (lipids, phenolics, nucleic acids, etc.). We propose here
three examples of protocols successfully tested in our laboratory on
different tree species (e.g., poplar, oak, willow, alder, eucalyptus). In
the three cases, the extracted proteins are coming from the entire
tissue considered (e.g., roots, stems, cambium, leaves). Protein
extraction methods using TCA/acetone precipitation, hot SDS,
and/or phenol extraction, respectively, are proposed [
9
-
11
]. We do
not consider here extraction as specifi c for compartments but rather
as a “total” cellular extraction. However, fractionation steps to iso-
late, e.g., chloroplasts, mitochondria, nuclei, or membranes [
12
-
14
], can precede these different methods. However, a protocol
should be optimized for individual studies. It is important to note
that depending on the physiological status of the plant tissue, proto-
cols may vary. Indeed, we observed that one protocol usually suit-
able for young or mature leaves was not optimal for senescent leaves.
So, optimization remains of uttermost importance.
The labelling reaction between the CyDyes and the proteins is
based on the formation of a covalent bond between the lysine resi-
dues of the proteins and the
N
-hydroxysuccinimidyl ester reactive
group. The CyDyes have been designed to have approximately the
same mass (around 500 Da) and to carry a positive charge to
replace the intrinsic positive charge of the lysine residue. This will
thus ensure that the pI of the protein is not modifi ed and its mass
barely changed, although it may be visible in the low molecular
mass range on the gel. Once the extraction has been optimized,
the proteins from different samples are quantifi ed, labelled, sepa-
rated, and compared (Fig.
2
) (
see
Note 3
).
Fluorescence and multiplexing can also be used to study phos-
pho- or glyco-proteins, namely, with Pro-Q
®
Diamond Phospho-
protein Stain or Pro-Q
®
Emerald Glycoprotein Stain from
Molecular Probes that can be used in combination with the Cydyes
or other fl uorescent staining (SyproRuby, LavaPurple).
Some other labels are now available on the market for protein
multiplexing, named G100, G200, and G300 developed by NH
