Abstract

ECHEVERRI, Nancy P; ORTIZ, Blanca L  and  CAMINOS, Jorge E. PROTEOMIC ANALYSIS OF PRIMARY CULTURES FROM THYROID. Rev.Colomb.Quim. [online]. 2010, vol.39, n.3, pp.343-358. ISSN 0120-2804.

In this paper, proteins of constitutive expression such as vimentin, actin, tubulin, heat shock protein of 60 kDa, peroxiredoxin and the mitochondrial ATP sintase were identified in primary cultures of normal thyroid and papillary carcinoma. The extraction conditions, solubilization, quantitative and qualitative analysis of such proteins were established. In addition, the best conditions for isoelectrofocusing (IEF) in the two-dimensional electrophoresis (2D) were founded. In the extraction and solubilization of proteins, the presence or absence ofampholytes or salts was evaluated. The best result in the extraction was obtained using buffer salts such as Tris-HCl and magnesium acetate, which increase the solubility of these proteins. For quantification, the recommendation is to combine colorimetric techniques with SDS-PAGE electrophoresis stained with Coomasie blue and Western blot to confirm the results, which allows verifying the integrity of these proteins. In the two-dimensional electrophoresis, resolved, focused and reproducible gels with greater number of spots were obtained using in the IEF immobilized pH gradients of 4-7 and final voltage of 8000 V. The proteins were identified by the bioinformatic analysis of the 2DE gels with the PDQuest program (PDQuest 7.2 (BioRad®) and MALDI-TOF.

Keywords : proteomics; isoelectrofocousing (IEF); 2D electrophoresis.

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