At4g16230 Antibody

What is At4g16230 and why is it important in plant research?

At4g16230 is a gene locus in Arabidopsis thaliana that encodes a GDSL esterase/lipase protein (O23470). This protein belongs to the GDSL family of lipolytic enzymes characterized by a distinct GDSL motif rather than the traditional GxSxG motif found in many lipases. The importance of GDSL esterase/lipase proteins in plant research stems from their diverse functional roles in plant development, stress responses, and secondary metabolism. The At4g16230-encoded protein specifically has been implicated in plant lipid metabolism and potentially in defense responses against pathogens.
Studies in Arabidopsis have shown that GDSL-family proteins can function in various physiological processes including seed development, pollen formation, and plant-pathogen interactions. The At4g16230 protein is part of this important family that contributes to Arabidopsis adaptation to environmental stresses and developmental regulation. Antibodies targeting this protein are valuable research tools for investigating its expression patterns, subcellular localization, and functional relationships with other biomolecules .

What are the technical specifications of commercially available At4g16230 antibodies?

The commercially available At4g16230 antibodies are typically monoclonal IgG antibodies produced in mouse hosts. According to product specifications, these antibodies target specific epitopes within the N-terminus of the O23470 protein (the product of At4g16230 gene). The antibodies are generated using synthetic peptides as immunogens, with peptide sequences derived from both N-terminal and C-terminal regions of the protein .
Key technical specifications include:

• Host species: Mouse

• Clonality: Monoclonal IgG

• Format: Typically supplied as lyophilized supernatant

• Target specificity: GDSL esterase/lipase At4g16230 (O23470)

• Primary application: Western blotting (AbInsure™ positive)

• ELISA titer: Approximately 10,000, corresponding to detection capability of approximately 1 ng of target protein in Western blot applications
  Standard antibody preparations contain approximately 0.2 mg of purified antibody material and should be stored at -20°C after reconstitution, with manufacturers recommending avoiding repeated freeze-thaw cycles to maintain antibody integrity and performance .

What applications are At4g16230 antibodies validated for?

At4g16230 antibodies have been primarily validated for Western blotting applications, with manufacturers recommending a starting dilution of 1:1000. In dot blot assays, these antibodies demonstrate the capability to detect between 0.01-1 ng of the corresponding immunogen peptide, indicating good sensitivity for the target protein .
While Western blotting represents the primary validated application, there is potential for these antibodies to be used in other immunological techniques common in plant molecular biology research, including:

• Immunoprecipitation (IP): Some antibody packages are specifically recommended for immunoprecipitation applications, allowing researchers to isolate At4g16230 protein complexes from plant cell extracts

• Immunohistochemistry (IHC): For tissue-level localization studies, though specific validation data may be limited

• Immunofluorescence (IF): For subcellular localization studies in fixed cells or tissue sections
  It is worth noting that unlike some other plant antibodies that have been extensively characterized across multiple applications (such as the GAPDH antibody that has been validated for WB, IHC, IF/ICC, FC, IP, and other techniques ), At4g16230 antibodies have a more focused application range currently centered on Western blotting detection .

What are the optimal sample preparation protocols for At4g16230 antibody in Western blotting?

For optimal Western blotting results with At4g16230 antibody, sample preparation should follow established protocols for plant protein extraction with modifications to preserve the integrity of membrane-associated lipases. Begin with 100-200 mg of fresh Arabidopsis tissue (leaves, seedlings, roots, or reproductive organs depending on research focus) and grind thoroughly in liquid nitrogen. Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and freshly added protease inhibitor cocktail.
When working specifically with lipases like At4g16230, consider these critical modifications:

• Include 5 mM EDTA to inhibit metal-dependent proteases that can degrade lipases

• Add 1 mM DTT or 5 mM β-mercaptoethanol to preserve enzymatic activity by preventing oxidation of thiol groups

• Keep samples consistently cold (0-4°C) throughout the extraction process to minimize protein degradation

• Centrifuge extracts at 15,000 × g for 15 minutes at 4°C to remove cellular debris
  For SDS-PAGE separation, load 10-20 μg of total protein extract per lane on a 12% polyacrylamide gel, as this concentration provides optimal resolution for the expected molecular weight of At4g16230 protein. After transfer to nitrocellulose or PVDF membrane, block with 5% non-fat dry milk in TBS-T for 1 hour at room temperature. For primary antibody incubation, use the recommended 1:1000 dilution of At4g16230 antibody in blocking buffer overnight at 4°C .

How can I validate the specificity of At4g16230 antibody in my experiments?

Validating antibody specificity is crucial for ensuring experimental rigor when working with At4g16230 antibody. Multiple complementary approaches should be employed to establish confidence in antibody performance:
First, include appropriate positive and negative controls in Western blot experiments. For positive controls, use recombinant At4g16230 protein if available, or wild-type Arabidopsis tissues known to express the protein. For negative controls, leverage either genetic resources (At4g16230 knockout mutants if available) or pre-absorption controls where the antibody is pre-incubated with excess immunizing peptide before use.
Second, perform knockout validation if knockout or knockdown lines are available. Compare protein detection between wild-type plants and plants with reduced or eliminated expression of At4g16230. The antibody signal should be absent or significantly reduced in knockout lines, confirming specificity for the target protein.
Third, verify the detected protein's molecular weight matches the predicted size of At4g16230 protein (approximately 36-40 kDa depending on post-translational modifications). Significant deviations from expected molecular weight may indicate cross-reactivity with other proteins.
Fourth, consider peptide competition assays where the antibody is pre-incubated with increasing concentrations of the immunizing peptide before Western blotting. The specific signal should diminish proportionally with increasing peptide concentration .
A comprehensive validation strategy similar to approaches used for other plant antibodies, such as those documented for GAPDH antibodies, will ensure experimental results are both reliable and reproducible .

What are the recommended storage conditions and handling practices for maximizing At4g16230 antibody lifespan?

To maximize the functional lifespan and maintain consistent performance of At4g16230 antibodies, adherence to proper storage and handling practices is essential. Upon receiving lyophilized antibody preparations, store the unopened vial at the recommended temperature (typically -20°C) until ready for reconstitution .
For reconstitution:

• Allow the lyophilized antibody to equilibrate to room temperature (approximately 30 minutes) before opening to prevent moisture condensation on the product

• Reconstitute with the recommended volume of sterile water or buffer as specified in the product documentation

• Mix gently by swirling or inversion rather than vortexing to prevent antibody denaturation

• Allow the solution to sit for 5-10 minutes at room temperature to ensure complete dissolution
  After reconstitution, divide the antibody solution into small single-use aliquots (typically 10-20 μl) to minimize freeze-thaw cycles. Each freeze-thaw event can reduce antibody activity by approximately 10-15%, so limiting these events is crucial for maintaining sensitivity. Store aliquots at -20°C and avoid storing in frost-free freezers where temperature fluctuations occur .
  For day-to-day handling:

• Always use clean pipette tips and sterile tubes when handling the antibody

• When removing an aliquot from storage, thaw it on ice and use immediately

• Avoid prolonged exposure to room temperature or light

• Centrifuge the antibody vial briefly before opening to collect liquid that may be trapped in the cap

• Document the date of reconstitution and number of freeze-thaw cycles for each aliquot
  Following these practices, properly maintained At4g16230 antibody preparations should retain activity for at least 12 months after reconstitution.

What are common causes of false positives/negatives when using At4g16230 antibody and how can they be addressed?

False positive and false negative results when using At4g16230 antibody can arise from multiple sources. Understanding these potential pitfalls and implementing corrective strategies is essential for generating reliable data.
Common causes of false positives:

• Cross-reactivity with related GDSL lipases: Arabidopsis contains multiple GDSL family proteins with sequence similarities. To address this, perform additional validation using knockout lines or mass spectrometry identification of detected bands.

• Non-specific binding due to excessive antibody concentration: The recommended 1:1000 dilution may need adjustment based on your specific experimental conditions. Perform a dilution series (1:500 to 1:5000) to determine optimal antibody concentration that maximizes specific signal while minimizing background.

• Insufficient blocking: Inadequate blocking can result in non-specific binding. Extend blocking time to 2 hours or overnight at 4°C, or test alternative blocking agents like 3-5% BSA.
  Common causes of false negatives:

• Low expression levels of target protein: At4g16230 may be expressed at low levels in certain tissues or developmental stages. Increase protein loading (up to 30-50 μg per lane) or enrich for membrane proteins using appropriate fractionation techniques.

• Epitope masking due to protein modifications: Post-translational modifications may interfere with antibody binding. Try denaturing conditions that might expose hidden epitopes, or test reducing/non-reducing conditions.

• Antibody degradation: Improperly stored antibodies lose activity. Always validate antibody performance using positive controls, and replace antibodies that show diminished activity.

• Inefficient protein transfer: GDSL lipases may transfer inefficiently during Western blotting. Optimize transfer conditions by increasing transfer time or using specialized buffers for hydrophobic proteins.
  When troubleshooting, systematically adjust one variable at a time and document all changes to experimental conditions. This methodical approach allows identification of optimal parameters for successful detection of At4g16230 protein .

How can I optimize immunoprecipitation protocols for At4g16230 antibody?

Optimizing immunoprecipitation (IP) for At4g16230 requires careful consideration of the protein's properties as a GDSL esterase/lipase, which may have hydrophobic regions and membrane associations. The following comprehensive protocol incorporates key modifications to standard IP procedures to maximize efficiency and specificity:

• Lysis buffer optimization: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5-1% NP-40 or 0.5% Triton X-100, 1 mM EDTA, 1 mM DTT, and protease inhibitor cocktail. This gentler detergent concentration helps maintain protein-protein interactions while efficiently extracting membrane-associated proteins.

• Pre-clearing strategy: Pre-clear lysates by incubating with Protein A/G beads (20 μl per 1 mg of total protein) for 1 hour at 4°C with gentle rotation before adding antibody. This reduces non-specific binding.

• Antibody amount optimization: For 1 mg of total protein extract, start with 2-4 μg of At4g16230 antibody (scaled from recommended IP applications of similar antibodies) . Perform a titration experiment (1, 2, 4, and 8 μg) to determine optimal antibody concentration.

• Incubation conditions: Incubate lysate with antibody overnight at 4°C with gentle rotation to maximize antigen-antibody interaction while minimizing protein degradation.

• Bead selection and handling: Use 30-50 μl of Protein G magnetic beads for mouse monoclonal antibodies. Magnetic beads provide gentler separation compared to centrifugation, potentially preserving weak protein-protein interactions.

• Washing optimization: Perform 4-5 washes with decreasing stringency: first wash with lysis buffer, followed by washes with lysis buffer containing reduced detergent concentration (0.1%), and final washes with detergent-free buffer.

• Elution conditions: For gentle elution that preserves protein interactions, use competitive elution with excess immunizing peptide. For denaturing elution, use 0.1 M glycine (pH 2.5) or SDS sample buffer heated to 95°C for 5 minutes.

• Controls: Always include a negative control using non-immune IgG from the same species, and when possible, include an IP from At4g16230 knockout tissue as additional specificity control.
  This optimized protocol addresses the specific challenges of immunoprecipitating plant lipases and should provide a strong starting point for successful IP experiments with At4g16230 antibody .

What quality control tests should be performed on new lots of At4g16230 antibody?

When receiving new lots of At4g16230 antibody, implementing rigorous quality control is essential to ensure experimental reproducibility. A comprehensive quality control workflow should include:
1. Physical inspection and documentation:

• Visually inspect the antibody vial for any physical abnormalities

• Document lot number, date received, and expiration date

• Record reconstitution date and conditions
  2. Functional validation assays:

• Western blot validation: Perform side-by-side Western blots comparing the new lot with a previously validated lot using the same Arabidopsis samples

• Sensitivity assessment: Create a dilution series of recombinant protein or positive control lysate (if available) to determine the detection limit

• Cross-reactivity evaluation: Test against known related proteins or knockout samples to confirm specificity
  3. Quantitative performance metrics:

• Signal-to-noise ratio comparison: Calculate and compare signal-to-background ratios between antibody lots

• Titration analysis: Perform antibody dilution series (1:500, 1:1000, 1:2000, 1:5000) to determine optimal working dilution for the new lot

• EC50 determination: If performing ELISA, calculate the half-maximal effective concentration and compare between lots
  4. Documentation and record-keeping:

• Create a detailed report for each antibody lot including all test results

• Maintain a laboratory database of antibody performance characteristics

• Document any lot-to-lot variations that might affect experimental interpretation
  A well-documented quality control procedure should detect any significant variations between antibody lots. If variations exceed 20% in any key performance metric, consider using the previous lot for ongoing experiments to maintain consistency. Alternatively, adjust protocols (antibody concentration, incubation times) to compensate for lot-to-lot differences .

How can At4g16230 antibody be used in studying plant stress responses?

At4g16230 antibody serves as a powerful tool for investigating the role of GDSL esterase/lipase in plant stress responses, particularly given the emerging evidence linking lipid metabolism to stress adaptation in plants. When designing experiments to study stress responses using this antibody, researchers should implement a multi-faceted approach combining protein expression analysis with functional studies.
For abiotic stress studies, expose Arabidopsis plants to controlled stress conditions (drought, salinity, temperature extremes, or nutrient limitation) using standardized protocols. Harvest tissue samples at multiple time points (0, 3, 6, 12, 24, 48, and 72 hours) post-stress induction to capture the dynamic regulation of At4g16230 protein. Prepare protein extracts from both stressed and control plants, then use Western blotting with At4g16230 antibody to quantify relative protein abundance changes. This temporal profiling can reveal whether At4g16230 protein levels are responsive to specific stresses, suggesting potential functional roles.
For biotic stress investigations, challenge Arabidopsis with pathogens such as Pseudomonas syringae or Botrytis cinerea, then analyze At4g16230 protein levels in infected versus uninfected tissues. Given that some GDSL lipases have been implicated in plant immunity, At4g16230 may show altered expression patterns during pathogen attack. Complement protein expression studies with subcellular localization experiments using immunofluorescence microscopy, as stress conditions might trigger relocalization of the protein to specific cellular compartments.
Additionally, combine antibody-based detection with enzymatic activity assays for GDSL lipases to correlate protein abundance with functional activity under stress conditions. This integrated approach provides insights into both the regulatory mechanisms controlling At4g16230 expression and its functional significance in stress adaptation pathways .

What approaches can be used to study protein-protein interactions involving At4g16230?

Investigating protein-protein interactions involving At4g16230 requires a strategic combination of complementary techniques that leverage the availability of specific antibodies. The following methodological approaches provide a comprehensive framework for identifying and characterizing interacting partners:
Co-immunoprecipitation (Co-IP) coupled with mass spectrometry: Start with optimized immunoprecipitation using At4g16230 antibody to pull down the target protein along with its interacting partners from Arabidopsis extracts. Process the immunoprecipitated material for mass spectrometry analysis to identify candidate interacting proteins. This approach provides an unbiased screen for novel interaction partners. For optimal results, perform biological replicates and include appropriate negative controls (IgG pulldowns and, if available, immunoprecipitations from At4g16230 knockout plants).
Bimolecular Fluorescence Complementation (BiFC): For candidate interactors identified through Co-IP/MS, validate the interactions in planta using BiFC. This technique involves creating fusion constructs of At4g16230 and candidate interactors with complementary fragments of a fluorescent protein (typically YFP). When expressed in plant cells, physical interaction between the proteins brings the fluorescent fragments together, restoring fluorescence that can be visualized by confocal microscopy.
Proximity-dependent biotin identification (BioID): For more challenging or transient interactions, fuse At4g16230 to a promiscuous biotin ligase (BirA*) and express this fusion protein in Arabidopsis. The BirA* moiety will biotinylate proteins in close proximity to At4g16230 in vivo. These biotinylated proteins can then be affinity-purified using streptavidin and identified by mass spectrometry.
Yeast two-hybrid screening: As a complementary approach, use At4g16230 as bait in yeast two-hybrid screens against Arabidopsis cDNA libraries to identify binary protein-protein interactions. Validate positive hits using the methods described above.
For all identified interactions, perform validation using reciprocal Co-IP experiments with antibodies against the interaction partners. Additionally, consider functional validation by analyzing phenotypes of plants where the interaction is disrupted through mutation or by expressing interaction-deficient variants of At4g16230 .

How can At4g16230 antibody be adapted for chromatin immunoprecipitation (ChIP) studies?

While At4g16230 encodes a GDSL esterase/lipase rather than a typical DNA-binding protein, recent research has revealed unexpected nuclear roles for some metabolic enzymes, making ChIP studies potentially relevant. Adapting At4g16230 antibody for chromatin immunoprecipitation requires specific protocol modifications to overcome challenges associated with non-canonical DNA-interacting proteins.
The standard ChIP protocol must be modified in several key aspects: First, optimize crosslinking conditions by testing both formaldehyde (1% for 10 minutes) and dual crosslinking approaches (1.5 mM EGS followed by 1% formaldehyde) to capture potentially weak or indirect DNA associations. Second, modify sonication conditions to optimize chromatin fragmentation to 200-500 bp fragments, with sonication efficiency verified by agarose gel electrophoresis. Third, increase antibody amounts to 5-10 μg per ChIP reaction (compared to the 2-4 μg typically used for transcription factors) to compensate for potentially lower DNA-binding affinity.
Critical validation steps include performing ChIP-qPCR on candidate genomic regions identified through preliminary experiments or predicted based on metabolic functions. Always include negative control regions (typically housekeeping genes) and use IgG antibody as a procedural control. For genome-wide analysis, consider ChIP-seq to identify all potential At4g16230 binding sites, but be prepared for more complex bioinformatic analysis to distinguish direct from indirect binding events.
To address potential skepticism about non-canonical DNA-protein interactions, implement orthogonal validation approaches. These include DNA pull-down assays using recombinant At4g16230 protein with candidate DNA sequences, reporter gene assays to test functional consequences of binding, and genetic experiments examining the effects of At4g16230 mutation on expression of putative target genes.
By carefully adapting standard ChIP protocols and implementing rigorous validation strategies, researchers can explore potential nuclear functions of At4g16230, potentially revealing novel regulatory roles beyond its established enzymatic activity .

How does At4g16230 antibody performance compare with antibodies for related GDSL family proteins?

When comparing the performance of At4g16230 antibody with antibodies targeting other GDSL family proteins in Arabidopsis, several distinct performance characteristics emerge. The Arabidopsis genome encodes approximately 108 GDSL esterase/lipase family members, presenting significant challenges for antibody specificity due to sequence similarities within conserved domains. This comparative analysis examines sensitivity, specificity, and applicability across experimental platforms.
The At4g16230 antibody demonstrates good specificity with minimal cross-reactivity to other GDSL family members, likely due to targeting of unique N-terminal epitopes rather than the conserved catalytic domains. This specificity advantage translates to cleaner Western blot results with reduced background compared to antibodies targeting more conserved regions of GDSL proteins . In contrast, antibodies against GDSL proteins like At5g33370 and At3g48460 often require additional validation steps to confirm target identity.
In terms of sensitivity, At4g16230 antibody exhibits detection capabilities comparable to other plant protein antibodies, with documented ability to detect approximately 1 ng of target protein in optimal Western blot conditions . This sensitivity is within the same range as the widely used GAPDH antibody standard (1:5000-1:40000 dilution range) .
For application versatility, At4g16230 antibody has been primarily validated for Western blotting, while some commercial antibodies against other GDSL family members offer broader application profiles including immunohistochemistry and immunofluorescence. This difference likely reflects varying degrees of validation rather than inherent antibody properties.
The following table summarizes key performance comparisons:

How can At4g16230 antibody be integrated with proteomics approaches?

Integrating At4g16230 antibody with modern proteomics techniques creates powerful research workflows for comprehensive characterization of this GDSL esterase/lipase and its functional networks. These integrated approaches extend beyond simple protein detection to enable complex functional studies that reveal both expression dynamics and protein interactions.
Immunoaffinity-based proteomics represents a primary integration strategy where At4g16230 antibody is used for selective enrichment prior to mass spectrometry analysis. Optimize immunoprecipitation protocols using the antibody conjugated to magnetic beads or agarose resin, then process the enriched samples for LC-MS/MS analysis. This approach facilitates identification of post-translational modifications on At4g16230 protein and characterization of its interacting partners. For maximum sensitivity, consider using sequential elution from immunoaffinity columns, first with a mild elution buffer to preserve protein-protein interactions, followed by stringent conditions to recover the target protein.
Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can be informed by immunoblotting results with At4g16230 antibody. First, use the antibody to identify conditions where the protein is expressed, then develop targeted mass spectrometry assays for precise quantification across experimental conditions. This combination provides both visualization (via immunoblotting) and accurate quantification (via targeted MS) of protein dynamics.
Spatial proteomics approaches can be enhanced by combining subcellular fractionation with At4g16230 antibody detection. Fractionate plant tissues into distinct subcellular compartments, then use the antibody to track the protein's distribution across fractions. Complement this with immunofluorescence microscopy to visualize subcellular localization, creating a comprehensive spatial profile of At4g16230 distribution.
For researchers with access to advanced proteomics facilities, consider proximity-dependent labeling approaches. Express At4g16230 fused to enzymes like BirA* or APEX2 in Arabidopsis, which will biotinylate or otherwise label proteins in close proximity to At4g16230 in vivo. Use the At4g16230 antibody to confirm expression of the fusion protein, then identify labeled proteins by mass spectrometry, revealing the spatial interactome of At4g16230 .

How might new antibody technologies enhance At4g16230 research?

Emerging antibody technologies offer significant potential to advance At4g16230 research beyond current methodological limitations. These innovations span from antibody engineering to novel detection platforms that can revolutionize how researchers study this GDSL esterase/lipase in Arabidopsis.
Single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies represent a promising frontier. Their small size (approximately 15 kDa compared to 150 kDa for conventional antibodies) enables access to epitopes in confined spaces, potentially revealing previously undetectable pools of At4g16230 in membrane microdomains or protein complexes. Additionally, nanobodies can be expressed intracellularly as "intrabodies" to track and potentially modulate At4g16230 function in living plant cells, opening new avenues for dynamic protein studies.
Proximity-dependent labeling approaches coupled with At4g16230-specific antibodies can revolutionize interactome mapping. By fusing enzymes like TurboID or APEX2 to anti-At4g16230 nanobodies, researchers can create antibody-enzyme conjugates that label proteins in close proximity to endogenous At4g16230 without requiring genetic modification of the target. This approach bridges traditional antibody applications with modern interactome mapping techniques.
Super-resolution microscopy optimized for plant tissue, when combined with highly specific At4g16230 antibodies, can reveal nanoscale distribution patterns previously unresolvable by conventional microscopy. Techniques like Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) can achieve resolution down to approximately 20 nm, potentially revealing functional microdomains or clustering behavior of At4g16230 protein in response to developmental or stress signals.
Antibody engineering approaches to enhance specificity and affinity through techniques like complementarity-determining region (CDR) grafting or affinity maturation can address current limitations in distinguishing highly similar GDSL family members. These engineered antibodies with enhanced properties could enable more precise quantification of At4g16230 in complex plant extracts.
Additionally, multiplexed imaging technologies using differently labeled antibodies against At4g16230 and interacting partners can enable simultaneous visualization of multiple proteins within the same sample, providing spatial context for functional relationships .

What are potential applications of At4g16230 antibody in cross-species studies?

The application of At4g16230 antibody across different plant species represents an valuable approach for comparative functional studies of GDSL esterase/lipases in plant evolution and adaptation. While developed against Arabidopsis thaliana protein, this antibody may recognize conserved epitopes in orthologous proteins from related species, enabling evolutionary and functional comparisons.
Cross-species reactivity assessment should follow a strategic approach starting with closely related Brassicaceae family members. The antibody likely shows strong reactivity with orthologs from species like Arabidopsis lyrata, Capsella rubella, and Brassica species due to high sequence conservation. More divergent species within eudicots such as tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), or soybean (Glycine max) may show reduced but potentially detectable cross-reactivity. Sequence alignment analysis of potential orthologs should guide species selection, focusing on conservation within the antibody's epitope region.
In cross-species applications, modified Western blotting protocols are essential. Increase primary antibody concentration (starting with 2-3× the concentration used for Arabidopsis), extend incubation times (overnight at 4°C), and use more sensitive detection systems like enhanced chemiluminescence. Always run Arabidopsis samples as positive controls alongside the test species for direct comparison.
For successful cross-species studies, address several methodological considerations:

• Optimize protein extraction for each species, as buffer components may require adjustment based on species-specific characteristics like secondary metabolite content

• Validate detected bands by mass spectrometry to confirm identity of cross-reacting proteins

• Consider epitope mapping to identify the specific region recognized by the antibody, which helps predict cross-reactivity based on sequence conservation

• Use genetic resources when available (e.g., GDSL lipase mutants in model species) to confirm specificity
  This cross-species application enables comparative studies of GDSL lipase expression patterns across development and in response to environmental stresses, potentially revealing evolutionary conservation or divergence in regulatory mechanisms. Additionally, it facilitates identification of species with naturally higher expression levels of GDSL lipases, which may serve as improved sources for biochemical and structural studies .

How might At4g16230 antibody contribute to understanding plant lipid metabolism pathways?

At4g16230 antibody serves as a crucial molecular tool for deciphering the complex networks of plant lipid metabolism, providing insights that extend beyond the immediate function of this GDSL esterase/lipase. By enabling specific detection and isolation of this enzyme, the antibody facilitates several research approaches that collectively advance our understanding of plant lipid pathways.
Subcellular localization studies using At4g16230 antibody for immunofluorescence microscopy can reveal the precise cellular compartments where this enzyme functions. Unlike transcriptional data or fluorescent protein fusions that may alter trafficking, antibody-based detection shows the native distribution of endogenous protein. This localization data provides context for metabolic function, particularly important for lipid metabolism which spans multiple organelles including the endoplasmic reticulum, lipid droplets, chloroplasts, and plasma membrane.
Temporal expression profiling across developmental stages and in response to environmental signals can be achieved through quantitative Western blotting with At4g16230 antibody. This approach reveals regulatory patterns that correlate with specific metabolic states or lipid remodeling events. For instance, expression changes during seed development might indicate roles in storage lipid metabolism, while stress-induced expression could suggest involvement in membrane lipid modifications during adaptation.
For pathway mapping, combine antibody-based protein detection with lipidomic analyses to correlate At4g16230 protein levels with specific lipid profiles. This integrated approach can identify substrate-product relationships by examining how altered enzyme levels (detected via immunoblotting) correlate with changes in specific lipid species (measured by mass spectrometry). Further, compare lipid profiles between wild-type and At4g16230 mutant lines while monitoring related enzyme expression using specific antibodies to identify potential compensatory or regulatory relationships within the lipid metabolic network.
To determine enzymatic properties, use the antibody for immunopurification of native At4g16230 protein from plant tissues, preserving post-translational modifications that may regulate activity. This native protein can then be used in enzymatic assays with various lipid substrates to determine specificity and kinetic parameters, providing functional insights that complement genetic approaches .