Ewelina Fic1
Sylwia Kedracka-Krok1
Urszula Jankowska1
Artur Pirog1
Marta DziedzickaWasylewska1,2
Research Article
Department of Physical
Biochemistry, Faculty of
Biochemistry, Biophysics and
Biotechnology, Jagiellonian
University, Cracow, Poland
2
Department of Pharmacology,
Institute of Pharmacology Polish
Academy of Sciences, Cracow,
Poland
Keywords:
IEF / Protein precipitation / Proteomics / Rat brain / SDS-PAGE
DOI 10.1002/elps.201000197
1 Introduction
The term proteomics refers to the analysis of all proteins in
a whole cell, including the description of co- and posttranslationally modified proteins and alternatively spliced
protein variants [1]. Proteomic methodologies have made a
great deal of progress in recent years with the development of
high-resolution 2-DE for protein separation and profiling,
high-sensitivity MS for determination of the molecular weight
of peptides which result from enzymatic cleavage of proteins,
and sequencing of secondary ions, and bioinformatic
approaches to protein identification using software databases.
Sample preparation is crucial for conducting reliable
proteomic analysis [2, 3]. Samples should have a highprotein concentration and be free of salt and other interfering components, such as detergents, nucleic acids, lipids,
etc [4, 5]. Precipitation is widely used for processing of
biological molecules such as proteins [6]. This procedure is
used to concentrate and fractionate the target molecule from
various contaminants. For example, in the biotechnology
industry, protein precipitation is used to eliminate
contaminants commonly contained in blood [7].
Sample preparation depends on the origin of the cells or
the tissue. The first step is usually homogenisation or
sonication followed by protein precipitation and solubilisa-
2.1 Tissues
The efficiency of various methods of protein precipitation
was investigated using four rat brain structures:
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E. Fic et al.
and the mixture was vortexed. One volume of 20% TCA was
then added, and the mixture was vortexed followed by
incubation for 1 h at 201C. After that, the samples were
centrifuged at 15 000 g for 15 min at 41C, 0.5 mL ice-cold
acetone containing 20 mM DTT was added and the mixture
was spun again at 15 000 g for 15 min at 41C. Finally, the
supernatant was removed and the pellet was air dried.
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E. Fic et al.
100
Amygdala
90
Precipitation Efficiency %
Frontal Cortex
80
Hippocampus
70
Striatum
60
50
40
30
20
10
0
chloroform/methanol
acetone
TCA/acetone
TCA
TCA (+ sonication)
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Table 1. Percentage of hippocampal protein recovery and number of protein spots after various precipitation proceduresa)
Precipitation method
Percentage of
recovery (%)
Number of spots
Chloroform/methanol
Acetone
10%TCA/acetone
10%TCA
10%TCA (1sonication)
270.98732.51
270.98732.51
270.98732.51
270.98732.51
324.17741.22
242.77756.95
186.15754.78
89.67714.83
68.21736.72
249.60759.22
88.52711.62
68.70717.81
33.0172.80
24.08711.55
77.00719.69
667
665
563
629
699
a) Values are the mean7standard deviation of five independent experiments. The number of spots was determined using ImageMaster
2D Platinum v6.0 software.
Figure 3. 2-DE IEF followed by SDS-PAGE. Analysis of proteins from rat hippocampus after various precipitation methods.
(A) Homogenate without precipitation, (B) TCA, (C) TCA/acetone, (D and F) chloroform/methanol, (E) and (G) acetone. Images
(AD) represent samples prepared with paper wicks, whereas (F and G) represent samples prepared without paper wicks. During IEF,
small rectangular wicks were placed at the anode and cathode ends of the IPG strips just beneath the electrodes. The wicks absorb
excess water, salts and proteins with pI values that lie outside the pH range of the IPG strip.
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4 Concluding remarks
The development of standardised, reproducible methods for
protein isolation from tissues offers great potential for the
discovery and validation of biomarkers.
The comparison of protein content in four supernatants
revealed that the TCA/acetone method produced the lowest
efficiency for protein precipitation. Reduced protein loss
(fewer proteins in supernatants) occurred with the methanol/chloroform and TCA methods. The acetone method
yielded the lowest loss of proteins, as determined by analysis
of the supernatant. The acetone method yielded high
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5 References
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[2] Hirano, M., Rakwal, R., Shibato, J., Agrawal, G. K., Jwa,
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[3] Shaw, M. M., Riederer, B. M., Proteomics 2003, 3,
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[4] Antonioli, P., Bachi, A., Fasoli, E., Righetti, P. G.,
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[5] Garcia-Rodriguez, S., Castilla, S. A., Machado, A., Ayala,
A., Biotechnol. Lett. 2003, 25, 18991903.
[6] Chen, Y. Y., Lin, S. Y., Yeh, Y. Y., Hsiao, H. H., Wu, C. Y.,
Chen, S. T., Wang, A. H. J., Electrophoresis 2005, 26,
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