At4g30620 Antibody

What is At4g30620 and why is it significant for plant research?

At4g30620 encodes a protein called STIC2-Like (STCL), which shares approximately 78% structural similarity with STIC2 including similar exon structure . STIC2 has been identified as a stromal protein that interacts with both ALB4 and ALB3, which are key components involved in thylakoid protein targeting pathways in chloroplasts . The significance of At4g30620 lies in its potential role in specialized protein transport mechanisms within plant chloroplasts, particularly in relation to thylakoid membrane organization. Understanding this protein's function can provide insights into fundamental aspects of chloroplast biogenesis and photosynthetic machinery assembly.

What are the basic specifications of At4g30620 antibody?

The At4g30620 antibody (Product Code: CSB-PA877301XA01DOA) is a rabbit-raised polyclonal antibody generated against recombinant Arabidopsis thaliana At4g30620 protein . It is specifically designed for research applications in Arabidopsis thaliana systems and has been tested for ELISA and Western Blot applications . The antibody is supplied in liquid form in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . It requires storage at -20°C or -80°C, with recommendations to avoid repeated freeze-thaw cycles . The antibody undergoes antigen affinity purification to ensure specificity for its target protein.

How should At4g30620 antibody be stored and handled for optimal performance?

For optimal performance, At4g30620 antibody should be stored at -20°C or -80°C immediately upon receipt . Researchers should aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles, which can degrade antibody performance. When working with the antibody, it should be thawed on ice and kept cold during experimental procedures. The preservative (0.03% Proclin 300) in the storage buffer helps maintain stability, but proper handling remains crucial for maintaining antibody integrity . For long-term storage planning, researchers should note that At4g30620 antibody has a typical shelf-life of 1-2 years when stored properly, though specific batch information should be consulted.

What are the validated applications for At4g30620 antibody?

The At4g30620 antibody has been validated for ELISA and Western Blot applications for the detection and identification of the At4g30620 antigen . For Western Blot applications, the antibody can detect the native protein in Arabidopsis thaliana plant tissues, particularly chloroplast-enriched fractions. When optimizing experimental procedures, researchers should consider that this polyclonal antibody may recognize multiple epitopes on the target protein, potentially enhancing detection sensitivity but requiring careful controls to confirm specificity. The antibody has not been specifically validated for immunoprecipitation, immunohistochemistry, or flow cytometry, though these applications may be possible with appropriate optimization.

How can researchers validate the specificity of At4g30620 antibody?

Validating antibody specificity is critical for ensuring experimental reliability. For At4g30620 antibody, several approaches are recommended:

• Knockout/knockdown verification: Using tissue from At4g30620 knockout or knockdown plants as negative controls can definitively confirm antibody specificity.

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide or recombinant At4g30620 protein should abolish or significantly reduce signal in immunoassays if the antibody is specific .

• Mass spectrometry validation: Immunoprecipitation followed by mass spectrometry analysis can identify proteins pulled down by the antibody, confirming whether At4g30620 is the primary target .

• Multiple antibody verification: Using alternative antibodies that target different epitopes of At4g30620 can provide additional confirmation of specificity .

It's important to note that antibodies may sometimes exhibit cross-reactivity with structurally similar proteins. The case study of the anti-glucocorticoid receptor antibody clone 5E4, which showed cross-reactivity with AMPD2 and TRIM28, demonstrates the importance of rigorous validation procedures .

What are the optimal conditions for using At4g30620 antibody in Western blot applications?

For optimal Western blot results with At4g30620 antibody, researchers should:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing protease inhibitors to prevent degradation. For chloroplast proteins like At4g30620, enrichment of chloroplast fractions may improve detection.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation of proteins in the expected molecular weight range of At4g30620.

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in standard transfer buffer.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature .

• Primary antibody incubation: Dilute At4g30620 antibody 1:1000 to 1:2000 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 to 1:10000 dilution for 1 hour at room temperature .

• Detection: Develop using enhanced chemiluminescence reagents and exposure to X-ray film or digital imaging systems.

These conditions may require optimization based on specific experimental requirements and equipment available.

How can At4g30620 antibody be used to investigate protein-protein interactions in chloroplast protein targeting pathways?

At4g30620 (STIC2-Like) is likely involved in protein targeting pathways similar to its homolog STIC2, which interacts with ALB4 and ALB3 in thylakoid protein targeting . To investigate these interactions:

• Co-immunoprecipitation: Use At4g30620 antibody for immunoprecipitation followed by Western blot analysis to detect potential interacting partners. This can be complemented with reciprocal co-IP using antibodies against suspected interaction partners.

• Proximity labeling approaches: Combine At4g30620 antibody with enzyme-mediated proximity labeling techniques (such as BioID or APEX) to identify proteins in close proximity to At4g30620 in vivo . This approach has been successfully used to map interactomes of chloroplast proteins like AtTic40, AtTrxM2, and AtPic1 .

• Immunofluorescence co-localization: Use At4g30620 antibody in combination with antibodies against known chloroplast protein targeting components to assess co-localization in situ.

• Split-protein complementation assays: Utilize DHFR* reporter-based protein fragment complementation assays to investigate specific interactions between At4g30620 and potential partners in organelle-specific contexts .

These approaches can help elucidate whether At4g30620, like STIC2, functions in specialized thylakoid protein targeting pathways and interacts with components such as ALB4 and ALB3.

What strategies can address potential cross-reactivity issues with At4g30620 antibody?

Cross-reactivity is a common challenge with antibodies, as demonstrated by the anti-glucocorticoid receptor antibody clone 5E4, which showed binding to unintended proteins AMPD2 and TRIM28 . To address potential cross-reactivity with At4g30620 antibody:

• Pre-adsorption controls: Pre-incubate the antibody with recombinant At4g30620 protein before use in experiments. If signals remain after pre-adsorption, this may indicate cross-reactivity .

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins recognized by the antibody. Comparing these results across different experimental conditions can help distinguish specific from non-specific interactions .

• Epitope mapping: Identify the specific epitope(s) recognized by the antibody and perform sequence similarity searches to identify potential cross-reactive proteins in Arabidopsis.

• Knockout controls: Use tissue from At4g30620 knockout plants to identify any remaining signals that would indicate cross-reactivity.

• Multiple antibody approach: Validate findings using alternative antibodies targeting different epitopes of At4g30620 to confirm that observed signals are truly specific to the target protein .

How can At4g30620 antibody be utilized in studying chloroplast protein transport defects?

The At4g30620 protein (STIC2-Like) may be functionally related to the STIC2 protein, which has been identified as a suppressor of the chloroplast protein import mutant tic40 . To investigate chloroplast protein transport defects:

• Comparative protein expression analysis: Use At4g30620 antibody to analyze protein levels in wild-type plants versus mutants with defects in chloroplast protein import machinery (such as tic40, tic110, or stic2 mutants).

• Subcellular fractionation studies: Combine At4g30620 antibody detection with chloroplast fractionation techniques to track the localization and abundance of At4g30620 protein in different chloroplast compartments (envelope, stroma, thylakoids) under various genetic backgrounds or stress conditions.

• Pulse-chase experiments: Employ At4g30620 antibody in pulse-chase experiments to monitor protein transport kinetics in wild-type versus mutant plants.

• Co-localization with transport machinery: Use At4g30620 antibody alongside antibodies against known components of chloroplast protein transport machinery (TIC/TOC complexes, cpSRP pathway components) to assess potential functional relationships.

This approach can help determine whether At4g30620, like its homolog STIC2, plays a role in specialized thylakoid protein targeting pathways and potentially interacts with the chloroplast protein import machinery.

What are common issues encountered with At4g30620 antibody and how can they be resolved?

When troubleshooting, researchers should systematically adjust one variable at a time and include appropriate positive and negative controls in each experiment.

How should researchers interpret conflicting results between At4g30620 antibody and other detection methods?

When faced with conflicting results between At4g30620 antibody detection and other methods (e.g., RNA expression, mass spectrometry, alternative antibodies), researchers should:

• Consider technical limitations: Each detection method has inherent limitations. Antibodies may have cross-reactivity issues , RNA levels may not correlate with protein abundance, and mass spectrometry may miss low-abundance proteins.

• Evaluate experimental conditions: Different methods may perform optimally under different conditions. For example, certain fixation methods may mask epitopes recognized by an antibody.

• Assess protein modifications: Post-translational modifications, alternative splicing, or protein processing may affect antibody recognition but might be detectable by other methods.

• Reconcile through additional validation: When conflicts arise, employ orthogonal approaches like genetic manipulation (overexpression, CRISPR knockout) to definitively establish protein identity and function.

• Consider biological context: Protein expression and localization may vary depending on tissue type, developmental stage, or environmental conditions, potentially explaining apparent discrepancies.

The case of the anti-glucocorticoid receptor antibody clone 5E4 highlights how widely used antibodies can show unexpected binding patterns, emphasizing the importance of rigorous validation even with established reagents .

How can researchers distinguish between specific and non-specific signals when using At4g30620 antibody?

Distinguishing specific from non-specific signals requires multiple control experiments:

• Knockout/knockdown controls: The most definitive control is to test the antibody on samples from plants lacking or significantly reduced in At4g30620 expression. Any signal in these samples indicates non-specific binding.

• Peptide competition: Pre-incubating the antibody with excess immunizing peptide or recombinant At4g30620 protein should eliminate specific signals while leaving non-specific binding intact .

• Isotype controls: Using an isotype-matched control antibody (rabbit IgG) can help identify non-specific binding due to the antibody class rather than its specific paratope.

• Sequential dilution analysis: Specific signals typically follow a predictable pattern of intensity reduction with antibody dilution, while non-specific binding may disappear abruptly or persist unpredictably.

• Cross-validation: Comparing results from the At4g30620 antibody with those obtained using alternative methods (such as tagged protein expression or mass spectrometry) can help confirm signal specificity.

• Signal localization: For microscopy applications, specific signal should localize to the expected subcellular compartment (chloroplasts for At4g30620), while non-specific signal may appear more broadly distributed.

How can At4g30620 antibody be used to investigate evolutionary conservation of chloroplast protein targeting pathways?

The evolutionary conservation of chloroplast protein targeting pathways can be investigated using At4g30620 antibody through several approaches:

• Cross-species reactivity testing: Evaluate whether the At4g30620 antibody recognizes homologous proteins in other plant species. This can provide insights into the conservation of protein structure across evolutionary distances.

• Comparative proteomics: Use At4g30620 antibody to immunoprecipitate the protein and its interactors from different plant species, followed by mass spectrometry analysis to identify conserved and divergent interaction partners.

• Functional complementation studies: Combine antibody detection with heterologous expression experiments to determine whether At4g30620 homologs from other species can functionally complement Arabidopsis mutants.

• Analysis in evolutionary model systems: Apply the antibody in studies of early land plants, algae, or evolutionary intermediates to track the emergence and specialization of this protein family during chloroplast evolution.

At4g30620's relationship to STIC2, which interacts with components like ALB4 that have evolved specialized functions from their bacterial ancestors , makes this a particularly interesting target for evolutionary studies of chloroplast protein transport mechanisms.

What insights can be gained by combining At4g30620 antibody detection with genetic suppressor analysis?

The discovery of STIC2 as a suppressor of the chloroplast protein import mutant tic40 suggests that At4g30620 (STIC2-Like) might play a related role in chloroplast protein homeostasis. Researchers can leverage this connection by:

• Genetic interaction mapping: Use At4g30620 antibody to analyze protein levels and localization in various genetic backgrounds, particularly in combination with mutations in known chloroplast protein import components (tic40, alb4, alb3).

• Suppressor screening analysis: In newly identified suppressors of chloroplast import mutants, use At4g30620 antibody to assess whether At4g30620 protein levels or localization are altered, potentially indicating compensatory mechanisms.

• Structure-function correlations: Compare protein detection patterns between wild-type and various point mutants to identify functionally important domains or residues in the At4g30620 protein.

• Pathway reconstitution: Use antibody detection in genetic complementation experiments to determine the minimal set of components needed to restore functional protein targeting in mutant backgrounds.

The study of STIC2 revealed its involvement with ALB4 in a specialized thylakoid protein targeting pathway that becomes disadvantageous to plants in the absence of Tic40 . Similar studies with At4g30620 might reveal parallel or complementary functions in chloroplast protein homeostasis.

What are the emerging applications for At4g30620 antibody in plant stress response research?

At4g30620 antibody can be valuable for investigating potential roles of this protein in plant stress responses, particularly those affecting chloroplast function. Future research directions may include:

• Stress-induced expression dynamics: Using At4g30620 antibody to track protein expression and localization changes under various stresses (light stress, temperature extremes, oxidative stress, drought) to determine if At4g30620 participates in stress adaptation mechanisms.

• Post-translational modification analysis: Investigating whether At4g30620 undergoes stress-induced post-translational modifications by analyzing mobility shifts or using modification-specific detection methods in conjunction with the antibody.

• Interactome remodeling: Applying proximity labeling approaches with At4g30620 antibody to identify stress-induced changes in its protein interaction network.

• Chloroplast redox regulation: Given that other chloroplast proteins like TrxM2 show regulatory functions involving cysteine oxidation-reduction reactions , investigating whether At4g30620 undergoes similar redox regulation using appropriate experimental designs.

Understanding how specialized chloroplast proteins like At4g30620 respond to environmental challenges can provide insights into plant stress adaptation mechanisms and potentially inform strategies for improving crop resilience.

How might technical advances in antibody development improve future At4g30620 research?

Future At4g30620 research could benefit from several technological advances in antibody development:

• Monoclonal antibody generation: Development of monoclonal antibodies against At4g30620 could provide more consistent and specific detection than current polyclonal reagents.

• Domain-specific antibodies: Creating antibodies targeting specific domains of At4g30620 would allow more precise functional studies and potentially distinguish between protein isoforms.

• Conformation-specific antibodies: Antibodies that recognize specific conformational states could help elucidate At4g30620's functional dynamics and potential regulatory mechanisms.

• Nanobodies/single-domain antibodies: Smaller antibody formats might provide better access to epitopes in complex structures like thylakoid membranes or protein complexes.

• Multiplexed detection systems: Development of antibody panels for simultaneous detection of At4g30620 and its interaction partners would facilitate more comprehensive analysis of protein targeting complexes.

• Antibody engineering approaches: FcγR-independent agonistic antibodies with engineered hinges, similar to those developed for 4-1BB , could potentially be adapted for plant research to manipulate protein function in vivo.