Abstract
The classical proteomics approach for the identification of allergen candidates consists on the separation of proteins by high-resolution two-dimensional electrophoresis (2-DE) with subsequent IgE immunoblotting and further analysis of IgE-reactive protein spots with mass spectrometry. In this approach at least two gels most be run. One gel is used for staining and the other is for immunoblotting by antibodies labeled with specific immunostains. Additional functional characterizations require either protein purification or 2-DE replicates and appear to be time- and reagent-consuming. Here we described a modified double one-dimensional electrophoresis (D1-DE) allowing the conversion of a protein spot previously visualized by 2-DE into an extended protein band. In D1-DE, the purity of the protein of interest is similar to 2-DE spots, but its abundance is many times higher than what can be found in a 2-DE single spot allowing many other functional analyses from a single D1-DE separation.
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References
Nony E, Le Mignon M, Brier S, Martelet A, Moingeon P (2016) Proteomics for allergy: from proteins to the patients. Curr Allergy Asthma Rep 16:64
Mousavi F, Majd A, Shahali Y, Ghahremaninejad F, Shoormasti RS, Pourpak Z (2017) Immunoproteomics of tree of heaven (Ailanthus atltissima) pollen allergens. J Proteome 154:94–101
Hoffmann-Sommergruber K (2016) Proteomics and its impact on food allergy diagnosis. EuPA Open Proteom 12:10–12
Tiotiu A, Brazdova A, Longé C, Gallet P, Morisset M, Leduc V et al (2016) Urtica dioica pollen allergy: clinical, biological, and allergomics analysis. Ann Allergy Asthma Immunol 117:527–534
Poncet P, Sénéchal H, Clement G, Purohit A, Sutra JP, Desvaux FX et al (2010) Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients. Allergy 65:571–580
D’Amato A, Bachi A, Fasoli E, Boschetti E, Peltre G, Sénéchal H et al (2010) In-depth exploration of Hevea brasiliensis latex proteome and “hidden allergens” via combinatorial peptide ligand libraries. J Proteome 73:1368–1380
Shahali Y, Sutra JP, Fasoli E, D’Amato A, Righetti PG, Futamura N et al (2012) Allergomic study of cypress pollen via combinatorial peptide ligand libraries. J Proteome 77:101–110
Shahali Y, Sutra JP, Peltre G, Charpin D, Sénéchal H, Poncet P (2010) IgE reactivity to common cypress (C. Sempervirens) pollen extracts: evidence for novel allergens. W Allergy Organ J 3:229–234
Shahali Y, Nicaise P, Brazdova A, Charpin D, Scala E, Mari A et al (2014) Complementarity between microarray and immunoblot for the comparative evaluation of IgE repertoire of French and Italian cypress pollen allergic patients. Folia Biol 60:192
Shahali Y, Sutra JP, Haddad I, Vinh J, Guilloux L, Peltre G et al (2012) Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis. Electrophoresis 33:462–469
Altland K, Silke R, Hackler R (1981) Demonstation of human prealbumin by double one-dimensional slab gel electrophoresis. Electrophoresis 2:148–155
Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99
Demeulemester C, Peltre G, Laurent M, Panheleux D, David B (1987) Cyanogen bromide-activated nitrocellulose membranes: a new tool for immunoprint techniques. Electrophoresis 8:71–73
Shahali Y, Sutra JP, Hilger C, Swiontek K, Haddad I, Vinh J et al (2017) Identification of a polygalacturonase (Cup s 2) as the major CCD-bearing allergen in Cupressus sempervirens pollen. Allergy 72:1806–1810
Sénéchal H, Šantrůček J, Melčová M, Svoboda P, Zídková J, Charpin D et al (2018) A new allergen family involved in pollen food-associated syndrome: Snakin/gibberellin-regulated proteins. J Allergy Clin Immunol 141:411–414
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Shahali, Y., Sénéchal, H., Poncet, P. (2019). Double One-Dimensional Electrophoresis (D1-DE) Adapted for Immunoproteomics. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8814-3_9
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DOI: https://doi.org/10.1007/978-1-4939-8814-3_9
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