Abstract

MARTINEZ, Ana P.; MARTINEZ, Sixta T.  and  ARDILA, Harold D.. Electrophoretic conditions for analysis of apoplastic proteins in stems and roots of carnation (Dianthus caryophyllus L.) for proteomic studies. Rev.Colomb.Quim. [online]. 2017, vol.46, n.2, pp.5-16. ISSN 0120-2804.  https://doi.org/10.15446/rev.colomb.quim.v46n2.62958.

In the present study a workflow, that can be applied to different plant species, to obtain a high quality apoplastic extract and for proteomic analysis is described. For that, using carnation roots and stems, some important parameters for the extraction of these proteins were evaluated. The best conditions for the infiltration were provided by using a 0.1 M sodium phosphate buffer pH 6.5, 50 mM sodium chloride and β -mercaptoethanol 0.1%, with 3 vacuum times at 70 kPa for 20 s and centrifuged at 4°C at 1,000 g for 20 min, and then a subsequent protein precipitation and concentration with the Trichloroacetic acid/acetone protocol. These conditions can be used to obtain a good quality extract for 2D electrophoretic analysis of apoplastic proteins. Strips with pH gradients between 5-8 and 3-10 were used to run isoelectric analysis for extracts obtained from apoplast in roots and stems, respectively. The conditions described can be used to perform studies focused on apoplastic proteins and its role in biological phenomena involving this plant species.

Keywords : Apoplastic fluid; Fusarium oxysporum f. sp dianthi; plant-pathogen; electrophoresis.

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