Recombinant Nicotiana tabacum 52 kDa cell wall protein
Product Specs
Target Background
Q&A
Experimental Design for Studying Recombinant Nicotiana tabacum 52 kDa Cell Wall Protein
Q: How can I design an experiment to study the expression and purification of recombinant Nicotiana tabacum 52 kDa cell wall protein in a laboratory setting?
A:
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Host Selection: Choose a suitable host for recombinant protein expression. Nicotiana tabacum is often used due to its high transient expression levels and low alkaloid content .
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Vector Construction: Design a plasmid vector with the gene encoding the 52 kDa protein, including appropriate promoters and terminators for plant expression.
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Transformation: Use Agrobacterium-mediated transformation or biolistics to introduce the plasmid into tobacco cells.
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Protein Purification: Employ techniques like affinity chromatography or gel filtration to isolate the recombinant protein.
Data Analysis and Contradiction Resolution
Q: How do I analyze proteomic data from experiments involving the recombinant Nicotiana tabacum 52 kDa cell wall protein, especially when encountering contradictory results?
A:
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Data Quality Control: Ensure that mass spectrometry and chromatography data are of high quality and properly calibrated.
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Statistical Analysis: Use statistical tools to identify differentially expressed proteins and validate results with multiple replicates.
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Literature Review: Compare findings with existing literature on similar proteins to contextualize results and resolve contradictions.
Advanced Research Questions: Protein Function and Localization
Q: What methods can be used to investigate the function and subcellular localization of the recombinant Nicotiana tabacum 52 kDa cell wall protein?
A:
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Subcellular Localization: Use fluorescent protein tagging (e.g., GFP) to visualize the protein's localization within plant cells.
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Functional Assays: Conduct biochemical assays to assess the protein's enzymatic activity or interactions with other proteins.
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Protein-Protein Interactions: Employ techniques like co-immunoprecipitation or yeast two-hybrid assays to identify interacting partners.
Methodological Considerations for Protein Expression
Q: How can I optimize the expression levels of the recombinant Nicotiana tabacum 52 kDa cell wall protein in tobacco plants?
A:
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Promoter Selection: Choose a strong promoter (e.g., CaMV 35S) to drive high expression levels.
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Cultivation Conditions: Optimize growth conditions such as temperature, light, and nutrient supply to enhance protein yield.
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Post-Translational Modifications: Consider the impact of PTMs on protein stability and function.
Comparative Proteomics
Q: How can I compare the proteome of Nicotiana tabacum expressing the recombinant 52 kDa protein with that of wild-type plants?
A:
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Proteome Analysis: Use techniques like iTRAQ or label-free quantification to compare protein abundance between transgenic and wild-type plants.
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Data Interpretation: Focus on identifying proteins with altered expression levels that may interact with or be influenced by the recombinant protein.
Example Data Table: Proteomic Analysis of Recombinant Nicotiana tabacum
| Protein ID | Function | Wild-Type Expression | Transgenic Expression |
|---|---|---|---|
| Protein A | Defence | 100 units | 150 units |
| Protein B | Metabolism | 50 units | 75 units |
| Protein C | Transport | 200 units | 250 units |
This table illustrates how proteomic data can be organized to compare expression levels between wild-type and transgenic plants.
Research Findings
Recent studies have highlighted the importance of proteomics in understanding plant responses to stress and the role of specific proteins in sieve elements of Nicotiana tabacum . The identification of sieve element-specific proteins has provided insights into plant defense mechanisms and developmental signaling . Additionally, Nicotiana tabacum is recognized for its efficiency in recombinant protein production, making it an ideal host for studying proteins like the 52 kDa cell wall protein .
