Biology Reference
In-Depth Information
and pollen [ 13 , 14 ]. As monomers, lipids can bind proteins, reducing
their solubility and changing the properties (pI and molecular
mass) used for their electrophoretic separation [ 15 ]. As supramo-
lecular assemblies (e.g., oil bodies), an excess of lipids can cause
insuffi cient detergency, and decreased effi ciency of the detergent as
solubilizing agent [ 15 ]. This problem can be overcome by scaling
up the separation, and therefore diluting the sample, or by means
of chemical delipidation using organic solvents such as chloroform
and acetone [ 16 ]. However, this method may lead to severe loss of
proteins either because some proteins are soluble in organic sol-
vents or because precipitated proteins do not properly resolubilize
after pelletizing [ 15 , 17 ].
Here, we describe a method for cleaning up protein extracts
prepared from lipid-rich plant tissues that is suitable for both 1-D
and 2-D electrophoresis and is compatible with downstream appli-
cations. We routinely use this method in our research with olive
pollen and seeds but it is also effective with other tissues and plants.
Delipidation of protein extracts is achieved in three steps by (1)
centrifugation and lipid pad removal, (2) fi ltration through a
Sephadex G25 matrix-containing column by gravity, and (3) TCA/
acetone precipitation prior to protein resolubilization. Lipid
removal following this method results in high-resolution and
reproducible 1-D and 2-D gels of proteins (Fig. 1 ).
2
Materials
Prepare all solution using ultrapure water (18 M
cm at 25 °C)
and analytical grade reagents. Use dust-free gloves and clean glass-
ware and equipment. Proceed as quickly as possible in order to
minimize the time of handling.
Ω
2.1 Tissue
Homogenization and
Protein Extraction
1. Liquid nitrogen.
2. Mortar and pestle ( see Note 1 ).
3. Extraction buffer: 0.05 M Tris-HCl, pH 7.4 ( see Note 2 ), 1 %
(v/v) Triton X-100, 4 % (w/v) 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS) ( see Note 3 ),
40 mM dithiothreitol (DTT), 5 % (w/v) polyvinylpolypyrrol-
idone (PVPP) ( see Note 4 ), and 10
L/mL plant protease
inhibitor cocktail (catalog number: P9599, Sigma-Aldrich
Corp., St. Louis, MO, USA) ( see Note 5 ).
4. Magnetic stirrer.
5. Refrigerated centrifuge 5810R (catalog number: 5810
000.017, Eppendorf, Hamburg, Germany) or similar,
equipped with a rotor for 2 mL microcentrifuge tubes.
6. Syringes (3 mL) with very fi ne needles (purchased at
drugstore).
μ
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