Simultaneous chloroplast, mitochondria isolation and mitochondrial protein preparation for two-dimensional electrophoresis analysis of Ice plant leaves under well watered and water-deficit stressed treatments
| Autor: | Hoang T. Kim Hong, Truong Thi Bich Phuong, John C. Cushman, Matthew D. Wheatley, Nguyen T. Thu Thuy |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Chloroplasts Blotting Western Mitochondrion 01 natural sciences Mitochondrial Proteins 03 medical and health sciences chemistry.chemical_compound Stress Physiological 010608 biotechnology Lysis buffer Electrophoresis Gel Two-Dimensional Plant Proteins 030304 developmental biology Mesembryanthemum 0303 health sciences Spots biology Leupeptin Mesembryanthemum crystallinum Water biology.organism_classification Mitochondria Plant Leaves Chloroplast Electrophoresis chemistry Biochemistry Electrophoresis Polyacrylamide Gel Percoll Biotechnology |
| Zdroj: | Protein Expression and Purification. 155:86-94 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2018.11.010 |
| Popis: | This paper presents a simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of Ice plant (Mesembryanthemum crystallinum) and mitochondrial protein preparation for two-dimensional electrophoresis (2DE) analysis under well watered and water -deficit stressed treatments. The washed chloroplasts and mitochondria were purified with Percoll gradients prepared using a Master flex R pump. The chloroplast and mitochondrial proteins were extracted in lysis buffer containing a protease inhibitor mix supplemented with 1 μM Leupeptin and 1 μM E64, followed by precipitation with ice-cold acetone. The protein contents were determined by an EZQ protein quantitation kit. The results show that chloroplast and mitochondria isolated from Ice plant leaves via this protocol have pure and intact. The shape of chloroplast and mitochondria observed by microscopy were clear and sharp. This procedure was employed for assessing the significant differences in mitochondrial protein expression patterns from the well watered and water-deficit stressed treatment leaves collected at dawn (6 a.m.) and dusk (6 p.m.). The results showed 71 and 20 differentially abundant spots between control and CAM for 6 a.m. and 6 p.m., respectively. In addition, 32 protein spots were differentially abundant for 6 a.m. control compared with 6 p.m. control, and 45 protein spots were differentially abundant for 6 a.m. CAM compared with 6 p.m. CAM. Spots that displayed differential abundance for control compared with CAM likely included proteins involved in mitochondrial processes necessary for CAM function. Through further analysis, these proteins will be identified and characterized in the near future using mass-spectrometry-based techniques. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect