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Methods to Concentrate Proteins for Protein Isolation, Proteomic, and Peptidomic Evaluation

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Detection of Blotted Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1314))

Abstract

In protein isolation, proteomic, or peptidomic procedures, protein solutions are often concentrated to remove solvents and undesirable molecules, to separate protein fractions or to increase protein concentrations. Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvent, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate, and three-phase partitioning. Solvents can be removed by passage through a semipermeable barrier where protein solutions are forced against the barrier in a centrifuge tube or with increased pressure concentrating protein in the remaining solution. The semipermeable barrier can be surrounded by a hygroscopic reagent to draw the solvent across the membrane. Proteins can be concentrated by freeze-drying (lyophilization). All these methods to concentrate proteins are discussed.

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References

  1. Scopes RK (1988) Protein purification: principles and practice. Springer-Verlag, New York

    Google Scholar 

  2. Dennison C (2003) A guide to protein isolation, 2nd edn. Kluwer Academic Publishers, Dordrecht

    Book  Google Scholar 

  3. Pohl T (1990) Concentrations of proteins and removal of solutes. In: Deutsher MP (ed) Methods in enzymology 182. Academic, London, pp 68–83

    Google Scholar 

  4. Righetti PG, Fasoli E, Boschetti E (2011) Combinatorial peptide ligand libraries: the conquest of the “hidden proteome” advances at great strides. Electrophoresis 32:960–966

    Article  CAS  PubMed  Google Scholar 

  5. Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R (2003) Optimization of protein precipitation upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B 785:263–275

    Article  CAS  Google Scholar 

  6. Jaing L, He L, Fountoulakis M (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J Chromator A 1023:3170320

    Google Scholar 

  7. Ingham KC (1990) Precipitation of proteins with polyethylene glycol. Guide to protein purification. In: Deutsher MP (ed) Methods in enzymology 120. Academic, London, pp 301–308

    Google Scholar 

  8. Polson A, Potgieter JF, Larier JF, Mears GEF, Joubert FJ (1964) The fractionation of protein mixtures by linear polymers of high molecular weight. Biochim Biophys Acta 82:463–475

    Article  CAS  PubMed  Google Scholar 

  9. Polson A, Coetzer T, Kruger J, von Maltzahn E, van der Merwe KJ (1985) Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol Invest 14:323–327

    Article  CAS  PubMed  Google Scholar 

  10. Goldring JPD, Coetzer THT (2003) Isolation of chicken immunoglobulins (IgY) from egg yolk. Biochem Mol Biol Ed 31:185–187

    Article  CAS  Google Scholar 

  11. Wessel D, Flugge UI (1984) A method for the qualitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143

    Article  CAS  PubMed  Google Scholar 

  12. Kay R, Barton C, Ratcliffe L, Matharoo-Ball B, Brown P, Roberts J, Teale P, Creaser C (2008) Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis. Rapid Commun Mass Spectrom 22:3255–3260

    Article  CAS  PubMed  Google Scholar 

  13. Koontz L (2014) TCA precipitation. Methods Enzymol 541:4–10

    Google Scholar 

  14. Goldring JPD, Brake DA, Cavacini LA, Long CA, Weidanz W (1989) Cloned T cells provide help for malaria specific polyclonal antibody responses. Eur J Immunol 19:559–561

    Article  CAS  PubMed  Google Scholar 

  15. Zhitkovich A, Costa M (1992) A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo. Carcinogenesis 13:1485–1489

    Article  CAS  PubMed  Google Scholar 

  16. Liu LF, Rowe TC, Liu Y, Tewey KM, Chen GL (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258:15365–15370

    CAS  PubMed  Google Scholar 

  17. Zhou J-Y, Dann GP, Shi T, Wang L, Gao X, Su D, Nicora CD, Shukla AK, Moore DJ, Liu T, Camp DG II, Smith RD, Qian W-J (2012) A simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomic applications. Anal Chem 84:2862–2867

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Goldring JPD, Ravaioli L (1996) Solubilization of protein dye complexes on nitrocellulose to quantify proteins spectrophotometrically. Anal Biochem 242:197–201

    Article  CAS  PubMed  Google Scholar 

  19. Dennison C, Lovrien R (1977) Three phase partitioning: concentration and purification of proteins. Protein Expr Purif 11:149–161

    Article  Google Scholar 

  20. Dennison C (2012) Three-phase partitioning. In: Tschesche H (ed) Methods in protein biochemistry. de Gruyter, Berlin

    Google Scholar 

  21. Ward W (2012) The art of protein purification. In: Ahmed R (ed). Protein purification

    Google Scholar 

  22. Pillay D, Boulange AF, Coetzer THT (2010) Expression, purification and characterization of two variant cysteine peptidases from Trypanosoma congolense with active site substitutions. Protein Expr Purif 74:264–271

    Article  CAS  PubMed  Google Scholar 

  23. Pike R, Dennison C (1989) Protein fractionation by three-phase partitioning (TPP) in aqueous/t-butanol/water/ammonium sulfate. Anal Biochem 180:169–171

    Article  PubMed  Google Scholar 

  24. Finoulst I, Pinkse M, Van Dongen W, Verheart P (2011) Sample preparation techniques for the untargeted LS-MS-based discovery of peptides in complex biological matrices. J Biomed Biotechnol 2011:245291

    Article  PubMed Central  PubMed  Google Scholar 

  25. Jennings A (1999) Lyophilization: introduction and basic principles. Taylor & Francis, Boca Raton

    Book  Google Scholar 

  26. Roy I, Gupta MN (2004) Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem 39:165–177

    Article  CAS  PubMed  Google Scholar 

  27. Schade R, Behn I, Erhard M, Hlinak A, Staak C (2001) Chicken egg yolk antibodies, production and application. Springer lab manual. Springer, Berlin

    Book  Google Scholar 

  28. Ning Z, Hawley B, Seebun D, Figeys D (2014) Apols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis. J Membr Biol 247:941–947

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Correspondence to J. P. Dean Goldring .

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Goldring, J.P.D. (2015). Methods to Concentrate Proteins for Protein Isolation, Proteomic, and Peptidomic Evaluation. In: Kurien, B., Scofield, R. (eds) Detection of Blotted Proteins. Methods in Molecular Biology, vol 1314. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2718-0_2

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  • DOI: https://doi.org/10.1007/978-1-4939-2718-0_2

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2717-3

  • Online ISBN: 978-1-4939-2718-0

  • eBook Packages: Springer Protocols

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