Abstract
Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds.
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Dr. J. Slovin and Dr. D. Lakshman are thanked for their critical review of this manuscript. Funding for this research was provided by ARS project 1275-21000-223-00D
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Natarajan, S.S., Xu, C., Bae, H. et al. Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS. Plant Mol Biol Rep 25, 55–62 (2007). https://doi.org/10.1007/s11105-007-0007-2
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DOI: https://doi.org/10.1007/s11105-007-0007-2