TPS21 Antibody

What is TPS21 and what is its function in plants?

TPS21 is a sesquiterpene synthase gene in Arabidopsis thaliana that encodes an enzyme responsible for converting farnesyl diphosphate into several sesquiterpene compounds, with (E)-β-caryophyllene being the major product. Additional minor products include α-humulene and α-copaene. TPS21 is predominantly expressed in floral tissues, including stigma, anthers, nectarines, and sepals, where it contributes to the production of virtually all floral volatile sesquiterpenes in Arabidopsis . These volatile compounds likely function in plant-insect interactions, potentially attracting pollinators or repelling herbivores. The regulation of TPS21 expression involves phytohormone signaling pathways, particularly jasmonate (JA) and gibberellin (GA), mediated through transcription factors such as MYC2 .

The sesquiterpenes produced by TPS21 contribute to the plant's chemical communication with its environment. Unlike some other terpene synthase genes that are expressed in roots or vegetative tissues, TPS21 shows a flower-specific expression pattern, suggesting its importance in reproductive biology and ecological interactions specific to floral structures .

What types of TPS21 antibodies are currently available for research?

Based on the available search results, polyclonal antibodies against TPS21 are commercially available for research purposes. For instance, there is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana TPS21 protein . This antibody is supplied in liquid form, preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . It is purified using antigen affinity methods, ensuring specificity for the target protein .

The antibody has been validated for several applications, including ELISA and Western blotting, which are essential techniques for detecting and quantifying TPS21 protein expression in plant tissues . Researchers should note that these antibodies are specifically designed for research use only and not for diagnostic or therapeutic procedures, making them appropriate tools for investigating TPS21 expression and function in academic research settings .

How do phytohormones regulate TPS21 expression?

TPS21 expression is significantly regulated by plant hormones, particularly gibberellin (GA) and jasmonate (JA). Both hormones can induce the expression of TPS21, leading to increased production of sesquiterpenes, especially (E)-β-caryophyllene . This regulatory mechanism requires the transcription factor MYC2, which directly binds to the promoter of TPS21 and activates its expression .

In the GA signaling pathway, DELLA proteins act as repressors. When GA levels are low, DELLA proteins accumulate and negatively affect sesquiterpene biosynthesis by repressing TPS21 expression. Experimental evidence has shown that TPS21 is downregulated in plants overexpressing REPRESSOR OF GA1-3 (RGA), one of the Arabidopsis DELLA proteins . Conversely, TPS21 is upregulated in penta DELLA-deficient mutants, confirming the negative regulation by DELLA proteins .

The JA signaling pathway involves the COI1/JAZs/MYC2 module, where JAZ proteins act similarly to DELLAs in GA signaling. MYC2 serves as a critical integration point between these two signaling pathways, as it directly interacts with DELLA proteins, specifically RGA, as demonstrated through yeast two-hybrid and coimmunoprecipitation assays . The N-terminus of MYC2 is responsible for binding to RGA in yeast cells . This interaction likely modulates MYC2's transcriptional activity on TPS21.

What are the optimal conditions for using TPS21 antibodies in Western blotting?

When using TPS21 antibodies for Western blotting, researchers should follow specific protocols to ensure optimal results. The antibody has been validated for Western blot (WB) applications to identify the TPS21 antigen . For optimal results, consider the following methodological approach:

• Sample Preparation: Extract total protein from Arabidopsis tissues (preferably floral tissues where TPS21 is predominantly expressed) using a buffer containing protease inhibitors to prevent protein degradation.

• Protein Separation: Use SDS-PAGE with 10-12% acrylamide gels for optimal separation of proteins in the molecular weight range of TPS21 (approximately 65-70 kDa).

• Transfer Conditions: Transfer proteins to PVDF or nitrocellulose membranes using standard wet or semi-dry transfer methods (25V for 2 hours or 100V for 1 hour).

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature to minimize non-specific binding.

• Antibody Dilution: Dilute the TPS21 antibody appropriately (typically 1:1000 to 1:5000) in blocking buffer and incubate overnight at 4°C. The exact optimal dilution should be determined empirically for each lot of antibody.

• Washing: Perform 3-5 washes with TBST, 5-10 minutes each, to remove unbound antibody.

• Secondary Antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (as TPS21 antibodies are typically raised in rabbits) at 1:5000-1:10000 dilution for 1 hour at room temperature .

• Detection: Use enhanced chemiluminescence (ECL) reagents for detection and expose to X-ray film or use a digital imaging system.

How should TPS21 antibodies be stored to maintain optimal activity?

Proper storage of TPS21 antibodies is crucial for maintaining their activity and specificity over time. Based on the product information, upon receipt, TPS21 antibodies should be stored at -20°C or -80°C . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity .

For antibodies supplied in liquid form with preservatives such as Proclin 300 and glycerol (as is the case with the TPS21 antibody mentioned in the search results), the glycerol acts as a cryoprotectant that helps prevent freeze-thaw damage . If working with the antibody regularly, it's advisable to prepare small aliquots for single use to minimize the number of freeze-thaw cycles for the main stock.

For short-term storage (up to 1 week), antibodies can be kept at 4°C, but for longer periods, freezing is recommended. When thawing frozen antibodies, allow them to thaw completely at cool room temperature or on ice, and mix gently by flicking or inverting the tube rather than vortexing, which can damage the antibody structure.

What controls should be included in experiments using TPS21 antibodies?

To ensure the reliability and validity of results when using TPS21 antibodies, researchers should include the following controls:

• Positive Control: Include protein extracts from tissues known to express TPS21, such as Arabidopsis flowers, particularly from organs where TPS21 is highly expressed (stigma, anthers, nectarines, and sepals) .

• Negative Control: Use protein extracts from a TPS21 knockout or knockdown plant, or from tissues where TPS21 is not expressed, such as vegetative tissues of Arabidopsis.

• Loading Control: Include detection of a housekeeping protein (e.g., actin, tubulin, or GAPDH) to normalize for variations in protein loading and transfer efficiency.

• Antibody Specificity Control: Pre-incubate the antibody with excess purified TPS21 antigen before use in the assay to demonstrate that binding is specifically blocked by the target protein.

• Secondary Antibody Control: Include a lane where the primary antibody is omitted but the secondary antibody is applied to identify any non-specific binding of the secondary antibody.

• Protein Ladder: Use a molecular weight marker to confirm that the detected band is at the expected size for TPS21 protein.

• Cross-Reactivity Controls: When studying related terpene synthases, include protein extracts from plants expressing other TPS family members to assess potential cross-reactivity of the antibody.

How can TPS21 antibodies be used to study MYC2-mediated transcriptional regulation?

TPS21 antibodies can be powerful tools for investigating the MYC2-mediated transcriptional regulation of terpene biosynthesis. Research has shown that MYC2, a basic helix-loop-helix transcription factor, directly binds to the promoters of sesquiterpene synthase genes, including TPS21, and activates their expression . This regulation is integrated with both GA and JA signaling pathways.

To study this regulatory mechanism, researchers can use TPS21 antibodies in the following experimental approaches:

• Chromatin Immunoprecipitation (ChIP) Assays: By combining TPS21 antibodies with ChIP techniques, researchers can identify proteins interacting with the TPS21 gene or its regulatory regions. This can help confirm the binding of MYC2 to the TPS21 promoter and potentially identify other transcription factors or regulatory proteins involved.

• Co-Immunoprecipitation (Co-IP): TPS21 antibodies can be used in Co-IP experiments to investigate protein-protein interactions between TPS21 and potential regulatory proteins, including components of the MYC2 transcriptional complex.

• Immunolocalization Studies: Using fluorescently labeled TPS21 antibodies for immunofluorescence microscopy, researchers can visualize the subcellular localization of TPS21 protein and potentially its co-localization with transcriptional regulators.

• Protein Expression Analysis in Mutants: By comparing TPS21 protein levels in wild-type plants versus myc2 mutants or plants with altered GA or JA signaling (such as DELLA mutants), researchers can quantitatively assess how these pathways affect TPS21 expression at the protein level .

What approaches can be used to study the role of TPS21 in plant-insect interactions?

TPS21 is involved in the production of volatile sesquiterpenes that likely play roles in plant-insect interactions. To investigate these ecological functions, researchers can employ TPS21 antibodies in combination with other techniques:

• Protein Expression Analysis Following Insect Damage: Using TPS21 antibodies in Western blotting or immunohistochemistry, researchers can monitor changes in TPS21 protein levels in response to herbivory or mechanical damage, providing insights into the timing and localization of the plant's defensive response.

• Correlation Studies: By correlating TPS21 protein levels (detected using the antibody) with volatile emissions (measured by gas chromatography-mass spectrometry) and insect behavior (assessed through bioassays), researchers can establish functional relationships between gene expression, metabolite production, and ecological outcomes.

• Genetic Manipulation Combined with Protein Analysis: In plants with altered TPS21 expression (overexpression lines or knockdowns), TPS21 antibodies can confirm the success of genetic manipulation at the protein level before proceeding with insect interaction studies.

• Tissue-Specific Expression Patterns: Immunohistochemistry with TPS21 antibodies can reveal the precise cellular and tissue localization of TPS21 protein in floral organs, potentially identifying specific structures involved in producing volatile compounds that attract pollinators or repel herbivores.

• Temporal Expression Studies: By analyzing TPS21 protein levels at different times of day or during different developmental stages using TPS21 antibodies, researchers can link temporal patterns of protein expression to diurnal rhythms in volatile emission that may optimize plant-insect interactions .

How can TPS21 antibodies contribute to understanding phytohormone crosstalk in terpene biosynthesis?

TPS21 expression is regulated by both GA and JA signaling pathways, making it an excellent model for studying phytohormone crosstalk. TPS21 antibodies can contribute to this research area through several approaches:

• Quantitative Protein Analysis Following Hormone Treatments: By treating plants with GA, JA, or combinations of these and other hormones, and then analyzing TPS21 protein levels using the antibody, researchers can determine how different hormones interact to regulate terpene biosynthesis at the protein level.

• Protein Expression in Signaling Mutants: TPS21 antibodies can be used to analyze protein levels in plants with mutations in hormone signaling components (such as DELLA proteins or JAZ proteins) to dissect the specific contributions of each pathway .

• Time-Course Studies: Using TPS21 antibodies in time-course experiments following hormone application can reveal the dynamics of protein induction and potential differences in the timing of responses to different hormones.

• Immunoprecipitation Combined with Mass Spectrometry: TPS21 antibodies can be used to pull down TPS21 protein complexes, which can then be analyzed by mass spectrometry to identify post-translational modifications that might be regulated by different hormones, providing mechanistic insights into how hormone signaling affects enzyme activity.

The research has demonstrated that DELLAs and JAZs both interact with MYC2 to regulate TPS21 expression, creating a nexus for GA and JA signal integration . TPS21 antibodies provide a means to study the downstream effects of this regulatory network at the protein level.

What are common causes of false results when using TPS21 antibodies?

When working with TPS21 antibodies, researchers may encounter several issues that could lead to false or misleading results:

• Cross-Reactivity: The TPS21 antibody might cross-react with other terpene synthases, especially those with high sequence homology. For example, TPS11, another sesquiterpene synthase in Arabidopsis, shares significant sequence similarity with TPS21 and might be recognized by the same antibody . To address this issue, always include appropriate controls and consider using knockout lines to confirm specificity.

• Protein Degradation: TPS enzymes can be susceptible to degradation during extraction, leading to multiple bands or reduced signal in Western blots. Use fresh plant material, keep samples cold during processing, and include protease inhibitors in extraction buffers.

• Post-Translational Modifications: TPS21 might undergo post-translational modifications that affect antibody recognition or cause shifts in apparent molecular weight. Consider using phosphatase treatments or other enzymatic treatments to investigate whether observed band shifts are due to modifications.

• Extraction Efficiency: Terpene synthases may be associated with membranes or present in specific subcellular compartments, affecting extraction efficiency. Optimize extraction protocols for different tissue types and consider testing different extraction buffers.

• Antibody Batch Variation: Different lots of polyclonal antibodies may have varying specificities and optimal working dilutions. Always validate new antibody batches against known positive and negative controls.

• Technical Artifacts: Issues such as air bubbles during transfer, incomplete blocking, or contamination can create artifacts in immunoblotting. Follow good laboratory practices and include appropriate technical controls.

How can researchers resolve inconsistent TPS21 detection across different plant tissues?

Inconsistent detection of TPS21 across different plant tissues can be addressed through the following approaches:

• Optimization for Tissue-Specific Extraction: Different plant tissues contain varying levels of compounds that may interfere with protein extraction or antibody binding. Develop and optimize tissue-specific extraction protocols that account for these differences.

• Tissue-Specific Loading Controls: Rather than using a single loading control for all tissues, identify and validate loading controls that show consistent expression across the specific tissues being compared.

• Protein Enrichment Techniques: For tissues with low TPS21 expression, consider using enrichment techniques such as immunoprecipitation before Western blotting to concentrate the target protein.

• Sample Normalization: Standardize sample preparation by normalizing to total protein content using methods such as Bradford assay before loading gels. Consider loading higher amounts of total protein from tissues with expected lower TPS21 expression.

• Signal Enhancement Methods: For tissues with low TPS21 expression, use more sensitive detection methods such as enhanced chemiluminescence substrates or fluorescent secondary antibodies with digital imaging.

• Validation with Alternative Methods: Complement antibody-based detection with other techniques such as RT-qPCR to confirm expression patterns at the mRNA level and resolve discrepancies.

• Consideration of Developmental Timing: TPS21 expression varies with developmental stage, particularly in floral tissues . Ensure that tissues are collected at comparable developmental stages when making comparisons.

How should researchers interpret contradictory results between TPS21 protein levels and volatile terpene emissions?

Discrepancies between TPS21 protein levels (detected by antibodies) and volatile terpene emissions may arise from several factors. Researchers should consider the following when interpreting such contradictory results:

• Post-Translational Regulation: TPS21 enzyme activity may be regulated post-translationally, meaning that protein abundance does not always correlate with enzyme activity. Investigate potential regulatory mechanisms such as phosphorylation or protein-protein interactions.

• Substrate Availability: Terpene production depends not only on enzyme levels but also on the availability of the substrate farnesyl diphosphate. Measure substrate levels or examine the expression of upstream biosynthetic enzymes.

• Compartmentalization: Spatial separation of the enzyme from its substrate or sequestration of products can affect volatile emissions independent of protein levels. Consider conducting subcellular localization studies.

• Volatile Collection Methodology: Technical limitations in volatile collection methods can lead to inconsistent results. Optimize volatile collection protocols and consider factors such as temperature, airflow, and timing.

• Additional Enzymatic Steps: Some terpenes may undergo further modifications after initial synthesis by TPS21, affecting the correlation between enzyme levels and final volatile profiles. Investigate potential downstream enzymatic conversions.

• Competitive Pathways: Farnesyl diphosphate can be channeled into multiple competing pathways. Examine the expression and activity of other enzymes that utilize this substrate.

• Integration with Other Data: Combine protein data with transcript analysis and metabolite profiling to get a more comprehensive picture of the biosynthetic pathway regulation.

How might TPS21 antibodies contribute to engineering terpene production in crop plants?

TPS21 antibodies could play valuable roles in engineering enhanced terpene production in crop plants for improved resistance to pests or for the production of valuable compounds:

• Validation of Transgene Expression: In plants engineered to express TPS21 from Arabidopsis, the antibody can confirm successful protein production and help optimize expression systems.

• Screening Transgenic Lines: TPS21 antibodies can be used to screen and select transgenic lines with optimal protein expression levels, correlating antibody-detected protein levels with terpene production capacities.

• Subcellular Targeting Studies: When engineering TPS21 expression with different subcellular targeting sequences, antibodies can confirm proper localization to the intended compartments to optimize enzyme access to substrates.

• Protein Stability Assessment: TPS21 antibodies can help evaluate protein stability in different genetic backgrounds or under various environmental conditions, informing strategies to enhance enzyme persistence in engineered plants.

• Structure-Function Analysis: By detecting modified versions of TPS21 with specific amino acid changes, antibodies can support structure-function studies aimed at enhancing catalytic efficiency or altering product specificity.

• Monitoring Environmental Responses: TPS21 antibodies can track how protein levels respond to environmental conditions in engineered plants, helping to optimize cultivation conditions for maximal terpene production.

What emerging technologies might enhance the utility of TPS21 antibodies in research?

Several emerging technologies could significantly expand the research applications of TPS21 antibodies:

• Single-Cell Proteomics: Advances in single-cell protein analysis could allow TPS21 antibodies to reveal cell-type-specific expression patterns in plant tissues, providing unprecedented spatial resolution of terpene biosynthetic capacity.

• CRISPR-Based Tagging: Combining CRISPR gene editing with antibody-based detection could enable the study of endogenous TPS21 tagged with epitopes or fluorescent proteins while maintaining native regulation.

• Microfluidic Antibody Arrays: Development of plant-specific microfluidic antibody arrays could allow high-throughput screening of TPS21 expression across multiple genetic backgrounds or environmental conditions.

• Antibody Engineering: Creating recombinant antibody fragments with enhanced specificity for TPS21 could improve detection sensitivity and reduce cross-reactivity with related terpene synthases.

• Proximity Labeling Techniques: Coupling TPS21 antibodies with proximity labeling enzymes could reveal the protein interaction network surrounding TPS21 in its native cellular context.

• Nanobody Development: Development of camelid single-domain antibodies (nanobodies) against TPS21 could provide improved access to epitopes in fixed tissues or in vivo imaging applications.

• Mass Cytometry Adaptation for Plants: Adapting mass cytometry techniques for plant cells using TPS21 antibodies could allow simultaneous measurement of multiple proteins in the terpene biosynthetic pathway at the single-cell level.