Volume: 3, 2024
3rd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland:
ENVIRONMENT – PLANT – ANIMAL – PRODUCT
Abstract number: F008
DOI: https://doi.org/10.24326/ICDSUPL3.F008
Published online: 24 April 2024
ICDSUPL, 3, F008 (2024)
Comparison of the efficiency of poultry meat protein separation. Capillary electrophoresis vs. SDS-PAGE
Piotr Klimowicz1*, Aneta Tomczak1, Magdalena Zielińska-Dawidziak1
1 Department of Food Biochemistry and Analysis, Faculty of Food Science and Nutrition, Poznan University of Life Sciences, Wojska Polskiego 28, 62-623 Poznan, Poland
* Corresponding author: piotr.klimowicz@up.poznan.pl
Abstract
The SDS-PAGE method, used for more than 50 years in protein analysis, separates proteins by size. Challenges are prompting a search for alternatives less expensive than 2D electrophoresis, and more precise than one-way separation, such as capillary electrophoresis (CGE). CGE is able to solve the limitations of traditional SDS-PAGE. Materials: Pectoral muscle of laying hens samples were collected from a feeding experiment, in which hens were fed with fodders containing various contents of lupine and soy for 4 weeks. Collected breast muscles were frozen and stored at –80°C. Before starting the analyses, the samples were homogenized, pooled and the leak was separated. Extraction and Quantification: Meat samples were extracted using phosphate-buffered saline (PBS) and quantified with a 2-D Quant kit (Cytiva, USA). SDS-PAGE: The extracted proteins were subjected to electrophoresis under denaturing conditions in 15% and 8% polyacrylamide gels using separation at constant 20mA per gel. The molecular weight marker in range 7 to 240 kDa was used [E3215-01, (EURx Poland)]. Gels after staining were documented using CLIQS software (TotalLabQuant, UK). Capillary Gel Electrophoresis: For CGE electrophoresis samples were prepared according to the Agilent High Sensitivity Protein 250 Kit procedure and analyzed by Agilent 2100 Bioanalyzer (both from Agilent Technologies, Germany). CGE methods allowed the identification of a larger number of protein fractions in the tested meat samples (about 35 compare to 20 detected with SDS-PAGE). Moreover, the CGE results suggested the occurrence of changes in the protein profile of breast muscle depending on the feed used for laying hens. The variability was observed mainly in the range 7 to 25 kDa and 100 to 240 kDa. CGE method is more suitable for the determination of protein fractions with lower concentrations (1 ng/μL–3 μg/μL) than SDS-PAGE (optimal separation at concentrations around is 7 μg of protein) and seems more suitable for quantification. CGE can perform the analysis in 30 minutes in one experiment, which is much faster than SDS-PAGE, which gave the comparable results after two analysis in 8% and 15% gels. In this study, several limitations has been found to the use of CGE, such as solvent selection, the cost of analysis, and the lack of further possibility of identification the selected protein fractions by Western-Blotting, mass spectrometry.
Keywords: SDS-PAGE, CGE, meat protein, pectoral muscle, laying hens
How to cite
P. Klimowicz, A. Tomczak, M. Zielińska-Dawidziak, 2024. Comparison of the efficiency of poultry meat protein separation. Capillary electrophoresis vs. SDS-PAGE. In: 3rd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland: Environment – Plant – Animal – Product. https://doi.org/10.24326/ICDSUPL3.F008