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Organelles Proteomics


Emerging Science

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2084-722X
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Effect of Triton X-100 and Dtt Concentrations on Wide Range Two-Dimensional Gel Electrophoresis of Tissue, Cell and Fluid Proteomes

Sébastien Charneau
  • Corresponding author
  • Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia-DF, Brazil
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/ Gabriel Costa Nunes da Cruz
  • Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia-DF, Brazil
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/ Camila Miranda Costa
  • Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia-DF, Brazil
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/ Marcelo Valle de Sousa
  • Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia-DF, Brazil
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/ Carlos André Ornelas
  • Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia-DF, Brazil
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Published Online: 2013-07-04 | DOI: https://doi.org/10.2478/orpr-2013-0002

Abstract

High-resolution separation by two-dimensional gel electrophoresis (2- DE) is still challenging due to the intrinsic behavior of proteins, principally throughout isoelectric focusing separation. It is often observed low resolution of proteins in the alkaline pH region when using wide range pH gradients. Herein, we show the effect of different concentrations of Triton X‑100 and DTT in the sample buffer on wide range pH (3-10) 2-DE profiles of three different biological samples as Trypanosoma cruzi cells, honey bee brain tissue and human saliva fluid. Higher resolution, number and intensity of spots were achieved when 85 mM DTT and 2.5% Triton X‑100 were employed for cell and tissue samples. No improvement was observed for fluid proteins, probably because water-soluble proteins do not require special conditions for extraction and prevention of precipitation during isoelectric focusing.

Keywords: 2D-PAGE; isoelectric focusing; reducing agent; detergent; protein solubilization

References

  • [1] O’Farrell P.H., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 1975, 250, 4007-4021Google Scholar

  • [2] Rabilloud T., Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains, Proteomics, 2002, 2, 3-10PubMedCrossrefGoogle Scholar

  • [3] Magalhaes A.D., Charneau S., Paba J., Guercio R.A., Teixeira A.R., Santana J.M., et al., Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages, Proteome Sci., 2008, 6, 24Web of ScienceCrossrefGoogle Scholar

  • [4] Rabilloud T., Valette C., Lawrence J.J., Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension, Electrophoresis, 1994, 15, 1552-1558CrossrefPubMedGoogle Scholar

  • [5] Mechin V., Consoli L., Le Guilloux M., Damerval C., An efficient solubilization buffer for plant proteins focused in immobilized pH gradients, Proteomics, 2003, 3, 1299-1302PubMedCrossrefGoogle Scholar

  • [6] Chinnasamy G., Rampitsch C., Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds, Biochim. Biophys. Acta, 2006, 1764, 641-644Google Scholar

  • [7] Hoving S., Voshol H., van Oostrum J., Towards high performance two-dimensional gel electrophoresis using ultrazoom gels, Electrophoresis, 2000, 21, 2617-2621PubMedCrossrefGoogle Scholar

  • [8] Paba J., Santana J.M., Teixeira A.R., Fontes W., Sousa M.V., Ricart C.A., Proteomic analysis of the human pathogen Trypanosoma cruzi, Proteomics, 2004, 4, 1052-1059CrossrefPubMedGoogle Scholar

  • [9] Guercio R.A., Shevchenko A., Lopez-Lozano J.L., Paba J., Sousa M.V., Ricart C.A., Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox, Proteome Sci., 2006, 4, 11CrossrefPubMedGoogle Scholar

  • [10] Charneau S., Junqueira M., Costa C.M., Pires D.L., Fernandes E.S., Bussacos A.C., et al., The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors, Int. J. Mass Spectrom., 2007, 268, 265-276Web of ScienceCrossrefGoogle Scholar

  • [11] Garcia L., Saraiva Garcia C.H., Calabria L.K., Cruz G.C.N., Sanchez Puentes A., Bao S.N., et al., Proteomic analysis of honey bee brain upon ontogenetic and behavioral development, J. Proteome Res., 2009, 8, 1464-1473Web of ScienceCrossrefGoogle Scholar

  • [12] Parodi-Talice A., Duran R., Arrambide N., Prieto V., Pineyro M.D., Pritsch O., et al., Proteome analysis of the causative agent of Chagas disease: Trypanosoma cruzi, Int. J. Parasitol., 2004, 34, 881-886Google Scholar

  • [13] Blum H., Beier H., Gross H.J., Improved silver staining of plant-proteins, RNA and DNA in polyacrilamide gels, Electrophoresis, 1987, 8, 93-99CrossrefGoogle Scholar

  • [14] Hoving S., Gerrits B., Voshol H., Muller D., Roberts R.C., van Oostrum J., Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients, Proteomics, 2002, 2, 127-134CrossrefPubMedGoogle Scholar

  • [15] Valcu C.M., Schlink K., Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples, Proteomics, 2006, 6, 1599-1605CrossrefPubMedGoogle Scholar

  • [16] Parodi-Talice A., Monteiro-Goes V., Arrambide N., Avila A.R., Duran R., Correa A., et al., Proteomic analysis of metacyclic trypomastigotes undergoing Trypanosoma cruzi metacyclogenesis, J. Mass Spectrom., 2007, 42, 1422-1432CrossrefWeb of ScienceGoogle Scholar

  • [17] Andrade H.M., Murta S.M., Chapeaurouge A., Perales J., Nirde P., Romanha A.J., Proteomic analysis of Trypanosoma cruzi resistance to Benznidazole, J. Proteome Res., 2008, 7, 2357-2367CrossrefWeb of ScienceGoogle Scholar

  • [18] Perez-Morales D., Lanz-Mendoza H., Hurtado G., Martinez- Espinosa R., Espinoza B., Proteomic analysis of Trypanosoma cruzi epimastigotes subjected to heat shock, J. Biomed. Biotechnol., 2012, ID 902803Web of ScienceGoogle Scholar

  • [19] Li J., Zhang L., Feng M., Zhang Z., Pan Y., Identification of the proteome composition occurring during the course of embryonic development of bees (Apis mellifera), Insect Mol. Biol., 2009, 18, 1-9Web of SciencePubMedCrossrefGoogle Scholar

  • [20] Zheng A., Li J., Begna D., Fang Y., Feng M., Song F., Proteomic analysis of honeybee (Apis mellifera L.) pupae head development, PLoS One, 2011, 6, e20428Google Scholar

  • [21] Begna D., Han B., Feng M., Fang Y., Li J., Differential expressions of nuclear proteomes between honeybee (Apis mellifera L.) Queen and Worker Larvae: a deep insight into caste pathway decisions, J. Proteome Res., 2012, 11, 1317-1329Web of ScienceCrossrefGoogle Scholar

  • [22] Woltedji D., Song F., Zhang L., Gala A., Han B., Feng M., et al., Western honeybee drones and workers (Apis mellifera ligustica) have different olfactory mechanisms than eastern honeybees (Apis cerana cerana), J. Proteome Res., 2012, 11, 4526-4540Web of ScienceCrossrefGoogle Scholar

  • [23] Gala A., Fang Y., Woltedji D., Zhang L., Han B., Feng M., et al., Changes of proteome and phosphoproteome trigger embryo-larva transition of honeybee worker (Apis mellifera ligustica), J. Proteomics, 2013, 78, 428-446 CrossrefWeb of ScienceGoogle Scholar

About the article

Received: 2013-03-20

Accepted: 2013-06-11

Published Online: 2013-07-04

Published in Print: 2014-01-01


Citation Information: Organelles Proteomics, ISSN (Online) 2084-722X, DOI: https://doi.org/10.2478/orpr-2013-0002.

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© 2013 Sébastien Charneau et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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