Preparation of mare's colostrum and milk sample for 2-DE separation without acetone precipitation
Aleksandra Cichy1, Alicja Dratwa-Chałupnik 1 , Weronika Medeńska1, Małgorzata Ożgo 1, Ryszard Pikuła 2
1Department of Physiology, Cytobiology and Proteomics, Faculty of Biotechnology and Animal Breeding, West Pomeranian University of Technology in Szczecin, Klemensa Janickiego 29, 71-270 Szczecin, Poland
2Department of Monogastric Animal Sciences, Laboratory of Horse Breeding and Animalotherapy, Faculty of Biotechnology and Animal Breeding, West Pomeranian University of Technology in Szczecin, Klemensa Janickiego 33, 71-270 Szczecin, Poland
- Acta Sci. Pol. Zootechnica, 19(3), 2020
Abstract. Before electrophoretic separation is performed, the samples must be dissolved in a lysis buffer (necessary to keep proteins dissolved and unbound during proteomic analyses during for a separation of proteins on polyacrylamide gels). The first step in preparing samples for proteomic analyses is their precipitation using e.g. acetone. The aim of precipitation is to obtain proteins from the sample and to remove the compounds interfering with 2-D electrophoresis. Due to difficulties in dissolving some colostrum and mare's milk samples in buffer lysis electrophoretic separation of this biological material was performed without acetone precipitation of proteins. To assess the effectiveness of the applied method, after two-dimensional separation of proteins (2-DE), the obtained gels were stained and archived. The preparation of mare's colostrum and milk samples for proteomic analyses, consisting of defatting, then precipitation of caseins and separation 2-DE, which was not preceded by precipitation of the samples with acetone, resulted in the loss of many protein spots which made it impossible to identify them later using the mass spectrometer.
Keywords: proteomic analyses, defatted, precipitation of caseins