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Evaluation of Sample Preparation Methods from Rice Seeds and Seedlings Suitable for Two-Dimensional Gel Electrophoresis

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Abstract

In a proteomic study, sample preparation is very important because it affects the quality of protein profiles on two-dimensional gel electrophoresis (2-DE). This study investigated the suitability of four protein extraction methods—direct lysis buffer extraction, trichloroacetic acid (TCA)/acetone precipitation, phenol extraction, and polyethylene glycol (PEG) fractionation—from rice seeds and seedlings (Oryza sativa L. ssp. indica cv. Khao Dawk Mali 105). The effectiveness of these methods was evaluated by the protein quantity and the 2-DE profiling quality. This included the number of protein spots, the consistency and uniqueness of protein spots, and their distribution in different ranges of pI and molecular weight (M r ). For protein quantity, the phenol and direct lysis extraction methods gave the highest protein yield in rice seeds and rice seedlings, respectively. However, in terms of the quality of 2-DE profiles, samples prepared by the TCA/acetone and phenol methods exhibited higher protein resolution and more spots than the protein profile derived from direct lysis extract. In addition, TCA/acetone method had the efficiency for high M r protein detection. PEG fractionation provided the best 2-DE pattern in terms of resolution, number of spots, minimal streaking, and reproducibility. Moreover, PEG fractionation was better for determining low M r basic proteins.

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Abbreviations

2-DE:

Two-dimensional gel electrophoresis

2-ME:

2-Mercaptoethanol

BSA:

Bovine serum albumin

CHAPS:

3-((3-Cholamidopropyl) dimethylammonio)-1-propane sulphonate

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

IEF:

Isoelectric focusing

IPG:

Immobilized pH gradient

M r :

Molecular weight

MS:

Mass spectrometry

NL:

Nonlinear

PAGE:

Polyacrylamide gel electrophoresis

PEG:

Polyethylene glycol

PF:

Pellet fraction

PMSF:

Phenylmethylsulfonyl fluoride

PVPP:

Polyvinylpolypyrrolidone

Rubisco:

Ribulose bisphosphate carboxylase/oxygenase

SD:

Standard deviation

SDS:

Sodium dodecyl sulfate

SF:

Supernatant fraction

TCA:

Trichloroacetic acid

Tris:

Tris (hydroxymethyl) aminomethane

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Acknowledgments

The authors are grateful to Assoc. Prof. Dr. Aphichart Vanavichit of the Rice Science Center and Rice Gene Discovery Unit, Kasetsart University, Thailand, for providing the rice seeds used in this research. This research was supported financially by the Center of Excellence for Innovation in Chemistry (PERCH-CIC) and by the Graduate School, Chiang Mai University.

Conflict of Interest

The authors have declared no conflict of interest.

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Authors and Affiliations

  1. Center of Excellence for Innovation in Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand

    Aphinya Wongpia & Sugunya Mahatheeranont

  2. Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand

    Aphinya Wongpia, Sugunya Mahatheeranont & Hataichanoke Niamsup

  3. Forensic Science Program, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

    Khemika Lomthaisong

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  1. Aphinya Wongpia
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Correspondence to Hataichanoke Niamsup.

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Wongpia, A., Mahatheeranont, S., Lomthaisong, K. et al. Evaluation of Sample Preparation Methods from Rice Seeds and Seedlings Suitable for Two-Dimensional Gel Electrophoresis. Appl Biochem Biotechnol 175, 1035–1051 (2015). https://doi.org/10.1007/s12010-014-1333-0

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