A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins
Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the lo...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-01-01
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| Series: | Agronomy |
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| Online Access: | https://www.mdpi.com/2073-4395/11/1/107 |
| _version_ | 1797414973818798080 |
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| author | Mahmudur Rahman Lei Liu Bronwyn J. Barkla |
| author_facet | Mahmudur Rahman Lei Liu Bronwyn J. Barkla |
| author_sort | Mahmudur Rahman |
| collection | DOAJ |
| description | Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of <i>Brassica rapa</i> R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE. |
| first_indexed | 2024-03-09T05:41:40Z |
| format | Article |
| id | doaj.art-de2338b5566141ffa3a4f530da239156 |
| institution | Directory Open Access Journal |
| issn | 2073-4395 |
| language | English |
| last_indexed | 2024-03-09T05:41:40Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Agronomy |
| spelling | doaj.art-de2338b5566141ffa3a4f530da2391562023-12-03T12:24:59ZengMDPI AGAgronomy2073-43952021-01-0111110710.3390/agronomy11010107A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage ProteinsMahmudur Rahman0Lei Liu1Bronwyn J. Barkla2Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, AustraliaSouthern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, AustraliaSouthern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, AustraliaRapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of <i>Brassica rapa</i> R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.https://www.mdpi.com/2073-4395/11/1/107protein extractionseed proteins<i>Brassica rapa</i>2S albumin like napins11/12S globulin like cruciferins1D SDS-PAGE |
| spellingShingle | Mahmudur Rahman Lei Liu Bronwyn J. Barkla A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins Agronomy protein extraction seed proteins <i>Brassica rapa</i> 2S albumin like napins 11/12S globulin like cruciferins 1D SDS-PAGE |
| title | A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins |
| title_full | A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins |
| title_fullStr | A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins |
| title_full_unstemmed | A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins |
| title_short | A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins |
| title_sort | single seed protein extraction protocol for characterizing brassica seed storage proteins |
| topic | protein extraction seed proteins <i>Brassica rapa</i> 2S albumin like napins 11/12S globulin like cruciferins 1D SDS-PAGE |
| url | https://www.mdpi.com/2073-4395/11/1/107 |
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