At2g03936 Antibody

What is the At2g03936 antibody and what organism does it target?

The At2g03936 antibody is a rabbit polyclonal antibody specifically developed to target and bind to the At2g03936 protein in Arabidopsis thaliana. This antibody was generated using a recombinant Arabidopsis thaliana At2g03936 protein as the immunogen, making it highly specific for plant research applications . The At2g03936 gene encodes a protein with the UniProt accession number Q2V4A6, which is part of the Arabidopsis genome located on chromosome 2 .

When conducting plant molecular biology research, it's essential to understand that this antibody has been specifically designed and validated for plant species reactivity, with primary focus on Arabidopsis thaliana systems. The polyclonal nature of this antibody means it can recognize multiple epitopes on the target protein, potentially providing stronger signal detection compared to monoclonal antibodies that recognize only a single epitope .

What applications is the At2g03936 antibody validated for?

The At2g03936 antibody has been specifically validated for two primary applications: Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These validations indicate the antibody's reliability in these particular experimental contexts.

For Western blotting applications, the antibody can be used to detect the target protein after separation by SDS-PAGE and transfer to a membrane. This application allows researchers to determine the molecular weight of the target protein and assess its expression levels in different plant tissues or under various experimental conditions. For ELISA applications, the antibody can quantitatively measure the concentration of the target protein in solution, providing a sensitive method for protein detection .

It's important to note that while these two applications have been validated, researchers may need to perform their own validation if they wish to use this antibody for other applications such as immunohistochemistry (IHC), immunofluorescence (IF), or chromatin immunoprecipitation (ChIP). Such validation would typically include positive and negative controls, as recommended in antibody validation guidelines .

What are the technical specifications of the At2g03936 antibody?

The At2g03936 antibody has the following technical specifications according to detailed product information:

The antibody package includes 10mg of purified antibody, 200μg of recombinant immunogen protein/peptide (which serves as a positive control), and 1ml of pre-immune serum . These components provide researchers with the necessary tools for experimental setup, including appropriate controls for validation.

The polyclonal nature of this antibody means it was produced by immunizing rabbits with the target antigen, resulting in a heterogeneous mixture of antibodies that recognize different epitopes on the target protein. This can be advantageous for detection of native proteins or in applications where signal amplification is desired .

What controls should be included when using the At2g03936 antibody?

When designing experiments with the At2g03936 antibody, including appropriate controls is essential for result validation and experimental rigor. The following controls should be incorporated:

First, utilize the pre-immune serum provided with the antibody package as a negative control. This serum was collected from the host animal (rabbit) before immunization and helps identify any non-specific binding . Additionally, include a negative tissue control from non-plant sources or from plant tissues known not to express the target protein.

For positive controls, the recombinant immunogen protein/peptide (200μg) supplied with the antibody can be used . This control confirms that the antibody can detect its intended target. When performing Western blot analysis, include molecular weight markers to verify that the detected protein band matches the expected size of the At2g03936 protein.

For specificity validation, conduct competitive inhibition tests as recommended in antibody validation guidelines. In these tests, pre-incubate the antibody with excess target antigen before application to demonstrate that binding is specifically inhibited . For quantitative applications like ELISA, implement a standard curve using purified target protein at known concentrations. This helps in accurate quantification and determining the detection limits of the assay.

Direct binding assays should always include both positive and negative antibody and antigen controls, with at least one isotype-matched, irrelevant (negative) control antibody tested for comparison .

What is the optimal protocol for using At2g03936 antibody in Western blotting?

For optimal Western blot results with the At2g03936 antibody, researchers should follow this methodological approach:

Sample preparation is critical. Extract total proteins from plant tissues using an appropriate buffer containing protease inhibitors to prevent degradation. Quantify protein concentration using Bradford or BCA assay to ensure equal loading. Prepare samples in reducing SDS-PAGE loading buffer and heat at 95°C for 5 minutes.

For electrophoresis, load 20-40μg of protein per lane on a 10-12% SDS-polyacrylamide gel depending on the expected molecular weight of the At2g03936 protein. Run the gel at 100-120V until sufficient separation is achieved. Transfer proteins to a PVDF or nitrocellulose membrane using semi-dry or wet transfer systems (25V for 1.5 hours or 30V overnight at 4°C).

During blocking and antibody incubation, block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Dilute the At2g03936 antibody (determine optimal dilution through titration, typically starting at 1:1000) in blocking buffer and incubate overnight at 4°C. Wash the membrane 3-4 times with TBST, 5 minutes each.

For detection, incubate with HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000-1:10000 dilution for 1 hour at room temperature. Wash 3-4 times with TBST, then develop using chemiluminescent substrate and detect signal using a digital imager or X-ray film . Always include positive and negative controls as described in section 2.1 to validate specificity.

The specificity of the results should be validated through the absence of signal in negative controls and presence of a band at the expected molecular weight in positive controls. If multiple bands appear, optimization of antibody concentration and washing conditions may be necessary .

How should the At2g03936 antibody be stored to maintain optimal activity?

Proper storage of the At2g03936 antibody is critical for maintaining its functionality and specificity over time. According to product specifications, the antibody should be stored at either -20°C or -80°C for long-term preservation .

When handling the antibody, aliquot it into smaller volumes upon receipt to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce antibody activity. Each aliquot should be sufficient for a single experiment to avoid repeated freeze-thaw cycles of the same tube. Label each aliquot with the antibody name, concentration, and date.

During shipping, the antibody is transported on blue ice as indicated in the product specifications . Upon receipt, immediately transfer to appropriate long-term storage conditions. Before each use, thaw an aliquot slowly on ice rather than at room temperature to preserve antibody structure and function.

Monitor the performance of the antibody over time by including consistent positive controls in your experiments. Any significant reduction in signal intensity may indicate degradation of the antibody, requiring a fresh aliquot or potentially a new lot of the antibody .

How can I assess the specificity and cross-reactivity of the At2g03936 antibody?

Assessing specificity and potential cross-reactivity of the At2g03936 antibody is crucial for generating reliable research data. A comprehensive approach includes several methodological strategies:

First, perform epitope mapping to determine precisely which region(s) of the At2g03936 protein the antibody recognizes. This can be done using peptide arrays or deletion mutants of the protein. Knowing the exact epitope(s) allows for in silico analysis to identify potentially similar sequences in other proteins that might lead to cross-reactivity .

Conduct Western blot analysis using tissues from multiple plant species, particularly those phylogenetically related to Arabidopsis thaliana. Compare the banding patterns to identify any non-specific binding. A highly specific antibody should yield a single band of the expected molecular weight in Arabidopsis samples and reduced or absent signals in distantly related species .

Implement competitive binding assays where the antibody is pre-incubated with excess purified target protein before application to the sample. Effective competition resulting in signal reduction confirms specificity for the target. Additionally, use tissues from knockout or knockdown plants lacking the At2g03936 gene as negative controls. The absence of signal in these tissues strongly supports antibody specificity .

For fine specificity assessment, use inhibition studies with defined structural components (like peptides representing different regions of the target protein) to characterize antibody specificity quantitatively. The inhibition of antibody binding by soluble antigen should be measured quantitatively to determine specificity parameters .

What approaches can be used to determine the optimal concentration of At2g03936 antibody for different applications?

Determining the optimal concentration of At2g03936 antibody for different applications requires systematic titration experiments tailored to each specific method. This methodological approach ensures reliable results while conserving valuable antibody resources.

For Western blotting optimization, prepare a dilution series of the antibody (typically 1:500, 1:1000, 1:2000, 1:5000, and 1:10000) and test against constant amounts of positive control samples. Evaluate the signal-to-noise ratio at each concentration to identify the dilution that provides clear specific bands with minimal background. Remember that optimal concentration may vary depending on the abundance of your target protein in different samples .

In ELISA applications, create a checkerboard titration by varying both antibody concentration and antigen concentration. Typically start with antibody dilutions ranging from 1:100 to 1:10000 and plot the resulting data to identify the dilution that provides the widest dynamic range and lowest background. Calculate the affinity constant (Ka) to quantitatively assess antibody-antigen interaction strength .

When optimizing new applications beyond the validated ELISA and WB, such as immunofluorescence or chromatin immunoprecipitation, more extensive titration may be necessary. Start with the manufacturer's recommendations or concentrations that worked for similar antibodies, then adjust based on empirical results.

Document all optimization experiments thoroughly, including lot numbers, experimental conditions, and quantitative measurements of signal intensity. This documentation facilitates reproducibility and troubleshooting. For quantitative applications, determine the limit of detection (LOD) and limit of quantification (LOQ) using standard curves with purified recombinant At2g03936 protein at known concentrations .

How can the At2g03936 antibody be used in co-immunoprecipitation studies to identify protein interactions?

Co-immunoprecipitation (Co-IP) using the At2g03936 antibody can elucidate protein-protein interactions involving the At2g03936 protein in Arabidopsis, though this application requires careful optimization as it extends beyond the validated applications of ELISA and Western blotting .

Begin by preparing plant lysates under non-denaturing conditions to preserve protein complexes. Extract proteins using a gentle lysis buffer (typically containing 20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40 or 0.5% Triton X-100) supplemented with protease inhibitors. Clarify lysates by centrifugation (14,000×g for 15 minutes at 4°C).

For antibody coupling, conjugate the At2g03936 antibody to protein A/G beads or magnetic beads using cross-linking reagents like dimethyl pimelimidate (DMP) to prevent antibody co-elution. Alternatively, use pre-coupled anti-rabbit IgG beads and add the At2g03936 antibody directly to the lysate. Incubate the lysate with antibody-coupled beads overnight at 4°C with gentle rotation.

After incubation, wash the beads 3-5 times with cold lysis buffer to remove non-specifically bound proteins. Elute the immunoprecipitated complexes using either low pH elution buffer or by boiling in SDS-PAGE sample buffer. Analyze the eluted proteins by SDS-PAGE followed by Western blotting or mass spectrometry to identify interaction partners .

Critical controls must include a negative control using pre-immune serum or an irrelevant rabbit IgG antibody processed identically to identify non-specific binding. Additionally, perform a reverse Co-IP using antibodies against potential interaction partners to confirm results. For novel interactions, validate findings using complementary techniques such as yeast two-hybrid assays or bimolecular fluorescence complementation .

For interaction specificity assessment, consider using competition with recombinant At2g03936 protein to demonstrate displacement of interaction partners, providing evidence for specific rather than non-specific associations .

What are common issues encountered when using At2g03936 antibody in Western blots and how can they be resolved?

Researchers working with the At2g03936 antibody in Western blot applications may encounter several technical challenges. Here are methodological approaches to address these common issues:

High background signal often results from insufficient blocking or washing. Increase blocking time to 2 hours or overnight with 5% BSA or milk in TBST, and extend wash steps to 10 minutes each with larger volumes of TBST. Additionally, try decreasing the primary and secondary antibody concentrations or adding 0.1-0.5% Tween-20 to the antibody dilution buffer to reduce non-specific binding .

When dealing with weak or absent signals, first check protein transfer efficiency using reversible staining (Ponceau S) immediately after transfer. If transfer is adequate, increase the amount of loaded protein (up to 50-75μg), extend primary antibody incubation time to overnight at 4°C, or utilize a more sensitive detection system like enhanced chemiluminescence reagents. Consider using fresh antibody aliquots as repeated freeze-thaw cycles may reduce antibody activity .

Multiple bands or unexpected band patterns can indicate cross-reactivity, protein degradation, or post-translational modifications. To address this, include protease inhibitors during extraction and sample preparation, and denature samples thoroughly before loading. Conduct peptide competition assays with the immunizing antigen to determine which bands represent specific binding .

If encountering inconsistent results between experiments, standardize protocols rigorously including sample preparation methods, gel percentage, transfer conditions, and detection parameters. Implement an internal loading control (like actin or tubulin) to normalize signals between samples and prepare larger batches of working solutions to use across multiple experiments .

Document all troubleshooting steps systematically, including experimental conditions and outcomes, to build a knowledge base for future optimization and to facilitate reproducibility in your research group .

How can I validate that my At2g03936 antibody is still active after extended storage?

Validating the activity of the At2g03936 antibody after extended storage is essential for experimental reliability. A systematic approach includes several complementary methods to assess antibody functionality.

First, perform a sensitivity comparison test by running parallel Western blots using the stored antibody alongside a positive control (either a fresh aliquot or the recombinant immunogen provided with the kit) . Compare signal intensity between the two conditions using identical protocols, protein amounts, and exposure times. A significant reduction in signal strength (>50%) with the stored antibody suggests deterioration.

Conduct titration curves to assess binding capacity. Compare the dose-response relationship of the stored antibody to previous data or manufacturer specifications. Shifts in EC50 values (the concentration producing half-maximal response) indicate potential activity loss. For ELISA applications, determine if there are changes in the standard curve parameters compared to when the antibody was fresh .

Evaluate specificity retention by testing the antibody against known positive and negative controls. The stored antibody should maintain the ability to discriminate between samples containing the target protein and those without it. Loss of specificity might be indicated by the appearance of non-specific bands in Western blots or increased background signal.

For quantitative assessment, calculate the binding activity index by comparing current signal intensity to the original baseline measurement at a standardized antibody concentration. Binding activity retention of ≥70% is generally considered acceptable for research applications .

What strategies can enhance signal detection when using At2g03936 antibody with low-abundance proteins?

Detecting low-abundance proteins with the At2g03936 antibody requires specialized methodological approaches to enhance sensitivity without compromising specificity. Several strategic modifications to standard protocols can significantly improve detection limits.

Optimize protein extraction by using specialized buffers containing chaotropic agents like urea (7-8M) or thiourea (2M) that enhance solubilization of membrane-associated or hydrophobic proteins. Include protease inhibitor cocktails to prevent degradation, and consider using fractionation techniques to enrich for cellular compartments where the target protein is predominantly expressed .

For Western blotting, implement protein enrichment techniques such as immunoprecipitation to concentrate the target protein before detection. Using high-sensitivity substrates like femto-chemiluminescent reagents can increase detection limits by 10-50 fold compared to standard ECL. Additionally, optimize transfer conditions by using PVDF membranes (which have higher protein binding capacity than nitrocellulose) and adding 0.05% SDS to the transfer buffer to improve elution of high molecular weight proteins .

When using ELISA, amplification systems such as biotin-streptavidin detection can significantly enhance sensitivity. Consider using a sandwich ELISA format with a capture antibody against a different epitope of the At2g03936 protein, which can improve both specificity and sensitivity compared to direct detection methods .

Extended incubation times at lower temperatures (4°C overnight or even 24-48 hours) for the primary antibody can improve binding kinetics for low-abundance targets. Similarly, signal amplification techniques like tyramide signal amplification (TSA) can provide 10-100 fold signal enhancement for immunodetection methods .

For quantitative analyses, use digital imaging systems with high dynamic range and integration capabilities rather than film-based detection, allowing for optimization of exposure times without saturation of stronger signals .

How can At2g03936 antibody be incorporated into studies of plant stress responses?

The At2g03936 antibody can be strategically incorporated into plant stress response studies to elucidate protein-level changes that may not be detectable at the transcriptional level. This approach provides valuable insights into post-transcriptional regulation mechanisms.

Design time-course experiments exposing Arabidopsis thaliana to various stress conditions (drought, salt, temperature extremes, pathogen infection) and collect tissue samples at defined intervals. Use the At2g03936 antibody in Western blot analysis to quantify protein expression changes over time, correlating these with physiological stress markers . This temporal profiling can reveal whether the At2g03936 protein participates in early response mechanisms or later adaptation processes.

Implement subcellular fractionation protocols prior to immunodetection to determine if stress conditions alter the protein's subcellular localization. Changes in localization pattern can indicate functional shifts in response to stress stimuli. For this application, the Western blot protocol may need optimization for each subcellular fraction due to different buffer compositions .

Combine antibody-based detection with phosphoproteomics approaches to investigate whether stress conditions trigger post-translational modifications of the At2g03936 protein. Modified forms can be detected as mobility shifts in Western blots or through specialized phospho-specific detection methods if phosphorylation is suspected .

For comparative studies across different plant species, test the cross-reactivity of the At2g03936 antibody with homologous proteins in related species to assess evolutionary conservation of stress response mechanisms. This approach requires careful validation of antibody specificity in each species examined .

Document protein abundance changes quantitatively using appropriate image analysis software, normalizing to stable reference proteins that maintain consistent expression under the stress conditions being studied. Statistical analysis should account for biological replicates to ensure reproducibility and significance of observed changes .

What considerations are important when using At2g03936 antibody for comparative studies across different plant tissues?

When conducting comparative studies across different plant tissues using the At2g03936 antibody, several methodological considerations must be addressed to ensure valid and reproducible results.

Tissue-specific protein extraction optimization is essential as different plant tissues vary significantly in their composition of interfering compounds. For example, leaves contain high levels of phenolics and photosynthetic proteins, while roots have different extracellular matrix components. Adapt extraction buffers for each tissue type, potentially including specific additives like polyvinylpolypyrrolidone (PVPP) for phenolic-rich tissues or higher concentrations of detergents for membrane-rich samples .

Implement rigorous protein quantification using methods resistant to plant-specific interfering compounds, such as the Bradford assay or amido black staining. Validate equal loading not only by total protein amount but also by using tissue-independent reference proteins like actin or GAPDH, verifying their consistent expression across the tissues being compared .

Account for tissue-specific post-translational modifications that may affect antibody recognition. The At2g03936 protein might undergo different modifications in different tissues, potentially affecting epitope accessibility. Consider using dephosphorylation treatments or other modification-removing approaches if differences in detection are observed .

For tissues with substantially different protein composition, optimize blocking conditions separately to minimize tissue-specific background. Test different blocking agents (BSA, milk, commercial blockers) for each tissue type to identify optimal signal-to-noise ratios .

When analyzing results, implement normalization strategies that account for tissue-specific variables. Calculate relative expression using consistent references, and consider presenting data as fold-changes relative to a reference tissue rather than absolute values. Statistical analysis should include biological replicates from multiple plant individuals to account for natural variation .

How can the At2g03936 antibody be used in conjunction with other molecular techniques for comprehensive protein characterization?

Integrating the At2g03936 antibody with complementary molecular techniques creates a powerful multidimensional approach for comprehensive protein characterization. This integrated methodology provides insights into expression, localization, interactions, and functional aspects of the target protein.

Combine antibody-based detection with transcriptomic analysis to correlate protein levels detected by Western blot or ELISA with corresponding mRNA abundance measured by RT-qPCR or RNA-Seq. This correlation (or lack thereof) can reveal post-transcriptional regulatory mechanisms affecting the At2g03936 protein . Discrepancies between transcript and protein levels may indicate regulation through translation efficiency or protein stability.

Implement the antibody in chromatin immunoprecipitation (ChIP) assays if the At2g03936 protein is suspected to have DNA-binding properties. While this application extends beyond the validated uses , careful optimization could enable identification of genomic binding sites, especially if combined with sequencing (ChIP-seq) to map interactions genome-wide .

For protein interaction studies, use the antibody in co-immunoprecipitation followed by mass spectrometry (IP-MS) to identify interaction partners. Cross-validate identified interactions using yeast two-hybrid or bimolecular fluorescence complementation assays. This multi-technique verification strengthens confidence in protein-protein interaction networks .

Investigate protein dynamics through pulse-chase experiments coupled with immunoprecipitation using the At2g03936 antibody to determine protein half-life and turnover rates under different conditions. This approach requires metabolic labeling of proteins and sequential sampling over time .

For functional studies, combine antibody-based protein depletion (immunodepletion) in cell-free systems with activity assays to correlate protein presence with specific biochemical functions. Additionally, compare protein detection in wild-type plants versus mutant lines with altered At2g03936 expression to establish structure-function relationships .

What future research directions can be enhanced using the At2g03936 antibody?

The At2g03936 antibody represents a valuable research tool that can facilitate several emerging research directions in plant molecular biology and biochemistry. As our understanding of plant systems advances, this antibody can contribute to multiple frontier areas of investigation.

Stress-responsive proteome dynamics studies can be significantly enhanced through temporal profiling of At2g03936 protein expression across diverse abiotic and biotic stress conditions. By quantitatively analyzing the protein's abundance changes in response to climate change-related stressors like drought, heat, and pathogen pressure, researchers can develop more comprehensive models of plant stress response networks . This approach becomes particularly powerful when integrated with systems biology platforms that incorporate transcriptomics, metabolomics, and phenotypic data.

The antibody can enable detailed investigation of organelle-specific proteome changes by combining subcellular fractionation with immunodetection. This approach could reveal spatial regulation of the At2g03936 protein during developmental transitions or stress responses, providing insights into compartmentalization as a regulatory mechanism in plant cells .

For evolutionary studies, the antibody might be used to assess conservation and divergence patterns of the At2g03936 protein across plant species, particularly if cross-reactivity can be established with homologous proteins in other plants. This comparative analysis could illuminate evolutionary adaptations in protein structure and function across plant lineages with different ecological adaptations .

Methodologically, the antibody can be adapted for emerging techniques such as proximity labeling approaches (BioID or APEX) to map protein interaction neighborhoods in living plant cells. By fusing enzymatic tags to the At2g03936 protein and using the antibody to verify expression and localization, researchers could identify both stable and transient interactors in their native cellular context .

As synthetic biology applications in plants advance, the antibody could serve as an essential tool for verifying expression and proper folding of engineered variants of the At2g03936 protein, facilitating structure-function studies and potential biotechnological applications .

What are the most important considerations for ensuring reproducibility when using At2g03936 antibody across different research groups?

Ensuring reproducibility when using the At2g03936 antibody across different research groups requires systematic attention to several critical factors throughout the experimental workflow.

Comprehensive antibody validation and reporting is foundational. Research groups should perform independent validation of antibody specificity using approaches like Western blotting against known positive and negative controls, even when using the same antibody lot. Document and share detailed validation protocols, including images of full blots showing all bands detected, not just the region of interest . Implement the use of RRID (Research Resource Identifier) standards when referencing the antibody in publications to ensure unambiguous identification.

Standardization of experimental protocols is essential. Develop and share detailed standard operating procedures (SOPs) that specify all parameters, including buffer compositions, incubation times and temperatures, and detection methods. For Western blotting, specify protein extraction methods, gel percentages, transfer conditions, blocking agents, antibody dilutions, and washing procedures. For ELISA, document plate types, coating conditions, and detection systems .

Sample preparation harmonization greatly influences results. Standardize plant growth conditions (light intensity, photoperiod, temperature, growth medium composition) and tissue collection protocols (time of day, plant age, tissue selection criteria). Document protein extraction methods in detail, including buffer components, tissue-to-buffer ratios, and handling procedures .

Implement robust quality control measures consistently. Use common reference samples that can be shared between laboratories as inter-lab controls. Include quantitative assessments of antibody performance using standard curves with recombinant protein . Establish acceptance criteria for experimental validity, such as signal-to-noise ratios or coefficient of variation thresholds.

Data sharing and collaborative validation strengthen reproducibility. Establish repositories for sharing raw data, including unprocessed Western blot images and ELISA readings. Consider multi-laboratory validation studies for critical findings, where independent groups replicate key experiments using aligned protocols. Implement digital lab notebooks to capture all experimental details, including environmental conditions and reagent lot numbers .

How should researchers address potential batch variations when using different lots of the At2g03936 antibody?

Addressing potential batch variations between different lots of the At2g03936 antibody requires a systematic approach to ensure experimental consistency and reliable data interpretation across studies.

Implement comprehensive lot qualification procedures when receiving a new antibody lot. Before using a new lot for experiments, conduct side-by-side comparison tests with the previous lot using identical samples and protocols. Perform titration curves to determine if the optimal working concentration has changed. Compare signal intensity, specificity (band pattern in Western blots), and background levels. Calculate a similarity index between lots based on quantitative parameters like EC50 values or signal-to-noise ratios .

Maintain reference samples specifically for lot comparison. Prepare large batches of positive control samples (plant tissues expressing At2g03936) and aliquot for long-term storage at -80°C. Use these consistent reference samples when testing each new lot to minimize variables other than the antibody itself .

Document lot-specific validation data comprehensively. Create detailed records for each lot, including validation experiments, optimal working dilutions, and any observed differences from previous lots. When significant differences are detected, develop lot-specific protocols with adjusted parameters (concentration, incubation time, blocking conditions) .

Consider bridging studies when transitioning between lots during ongoing research projects. Run key experiments with both the old and new lot to establish correlation factors if quantitative differences are observed. Apply mathematical normalization based on these correlation factors when comparing data obtained with different lots .

For critical research findings, verify results with alternative detection methods independent of the At2g03936 antibody. These might include mass spectrometry-based protein identification, RNA expression analysis, or functional assays specific to the protein's activity .