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Talanta. 2014 Feb;119:90-7. doi: 10.1016/j.talanta.2013.10.019. Epub 2013 Nov 5.

A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples.

Author information

1
BIOSCOPE group, REQUIMTE, Department of Chemistry, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal; Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
2
BIOSCOPE group, REQUIMTE, Department of Chemistry, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal.
3
Servicio de Nefrología, Complejo Hospitalario Universitario de Ourense, Ourense, España.
4
Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
5
BIOSCOPE group, REQUIMTE, Department of Chemistry, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal; Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. Electronic address: hmsantos@fct.unl.pt.

Abstract

Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (2(4)) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p < 0.05) in protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop(®) technology, protein ABS at 280 nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified.

KEYWORDS:

Formalin-fixed; Protein extraction; Tissue proteomics; Ultrasonic; Ultrasound

PMID:
24401389
DOI:
10.1016/j.talanta.2013.10.019
[Indexed for MEDLINE]

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