Biology Reference
In-Depth Information
In plant research, 2D DIGE was applied successfully for analyses
of protein samples from various species and different tissues. For
instance, the technique was used to investigate different plant
developmental processes and organogenesis (
2-4
), plant organelle
proteomes (
7, 8
), analyses
of plants in associations to symbiotic and/or pathogenic microbes
(
9-13
), and posttranslational protein modifi cations in plants (
14
).
Due to the presence of a cell wall and very special biochemical
properties, disruption of plant cells and isolation of protein fractions
are challenging but of major importance to obtain high-resolution
gels. To fi nd a very effi cient protein purifi cation technique, four
different protein extraction protocols were tested in our laboratory:
(1) a rapid protocol without precipitation as described by Gallardo
et al. (
5, 6
), plants under abiotic stress conditions (
15
), (2) a protocol utilizing TCA precipitation in combination
with acetone (
16
), (3) a protocol described by Corcke and Roberts
(
17
) which relies on boiling of protein fractions in Laemmli buffer
(
18
) and subsequent protein precipitated with acetone, and (4) an
extraction method using phenol which is combined with an ammo-
nium acetate in methanol precipitation as described by Hurkman
and Tanaka (
19
), modifi ed by Colditz et al. (
20
). Optimal protein
purifi cation and resolution on IEF-SDS gels were achieved using the
modifi ed Hurkman and Tanaka (
19
) protocol even for low amounts
of tissue.
2. Materials
All buffers, solutions, and reagents are given in the order of usage
according to the methods in Subheading
3
. Prepare all solutions
freshly using analytical grade chemicals in combination with pure
deionized water.
2.1. Protein Extraction
with Phenol
1.
Extraction buffer: 700 mM sucrose, 500 mM Tris, 50 mM
EDTA, 100 mM KCl, pH 8.0 (HCl). Directly before usage,
2%
-mercaptoethanol is added. Extraction buffer is stored at
4°C or frozen at −20°C in aliquots.
β
2.
Water-saturated phenol (pH 6.6/7.9; Amresco, Solon, USA),
stored at 4°C.
3.
Precipitation solution: 0.1 M ammonium acetate in methanol,
stored at 4°C.
1.
Dithiothreitol (DTT).
2.2. DIGE Sample
Preparation
2.
Lysis buffer: 8 M urea, 4% [w/v] CHAPS, 40 mM Tris base.
Lysis buffer is stored at −20°C (see Note 1).
3.
CyDye™ fl uor minimal labeling reagents: Cy2™, Cy3™, and
Cy5™ (GE Healthcare, Munich, Germany). The fl uorophores
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