At4g39290 Antibody

What is the At4g39290 gene and why are antibodies against it important for research?

At4g39290 is a gene in Arabidopsis thaliana that encodes a protein involved in cellular stress response mechanisms, particularly under abiotic stress conditions. Antibodies targeting this protein are crucial for researchers investigating plant stress physiology, protein localization, and protein-protein interactions in Arabidopsis and related species. These antibodies enable direct visualization and quantification of At4g39290 protein expression patterns across different tissues and under various experimental conditions . The importance of these antibodies lies in their ability to track protein expression changes that may not correlate directly with transcriptional alterations, providing insights into post-transcriptional and post-translational regulatory mechanisms that affect stress response pathways in plants. Most commercially available At4g39290 antibodies target specific epitopes within the protein's functional domains, allowing researchers to investigate domain-specific functions and interactions with other cellular components.

How do I validate the specificity of an At4g39290 antibody for my experiments?

Validating antibody specificity is critical for ensuring experimental reliability when working with At4g39290. Begin with Western blot analysis using wild-type Arabidopsis samples alongside At4g39290 knockout or knockdown lines. A specific antibody will show reduced or absent signal in the knockout/knockdown samples compared to wild-type. For more rigorous validation, perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide before application to Western blots or immunostaining procedures . Additionally, immunoprecipitation followed by mass spectrometry analysis can confirm that the antibody is truly capturing the At4g39290 protein rather than cross-reacting with other proteins. Cross-reactivity testing against related plant species can also help establish specificity boundaries. Document band sizes carefully, as post-translational modifications may result in size shifts from the predicted molecular weight. Finally, compare results with antibodies targeting different epitopes of the same protein, as concordant results across different antibodies strengthen confidence in specificity.

What protocols are recommended for immunolocalization of At4g39290 in plant tissue samples?

For optimal immunolocalization of At4g39290 in plant tissues, consider the following methodological approach: First, fix tissue samples in 4% paraformaldehyde for 2-4 hours at room temperature, followed by embedding in either paraffin for thin sectioning or agarose for vibratome sectioning, depending on the required resolution. For whole-mount preparations of Arabidopsis seedlings, extend fixation time to ensure complete penetration. Blocking should be performed with 3-5% BSA containing 0.1% Triton X-100 for at least 1 hour at room temperature . Primary antibody incubation with At4g39290 antibody (typically at 1:250-1:500 dilution) should proceed overnight at 4°C, followed by thorough washing in PBS with 0.1% Tween-20. For detection, secondary antibodies conjugated with bright fluorophores like Alexa Fluor 488 or 594 typically yield the best results. When analyzing subcellular localization, co-staining with organelle markers (such as DAPI for nuclei or MitoTracker for mitochondria) is strongly recommended. For tissues with high autofluorescence, consider implementing spectral unmixing during confocal microscopy or using Near-IR fluorophores to improve signal-to-noise ratios. Always include negative controls (secondary antibody only) and wild-type versus knockout comparisons to validate staining specificity.

How can I optimize Western blot conditions for detecting low-abundance At4g39290 protein?

Detecting low-abundance At4g39290 protein requires systematic optimization of multiple Western blot parameters. Begin by enriching the target protein through subcellular fractionation, focusing on the organelle fraction where At4g39290 is predominantly localized. Tissue selection is critical—prioritize tissues or developmental stages with known higher expression based on transcriptomic data . For protein extraction, incorporate protease inhibitor cocktails supplemented with specific inhibitors like PMSF (1 mM) and phosphatase inhibitors if phosphorylated forms are of interest. Optimize protein loading to 50-75 μg per lane, and consider using gradient gels (4-15%) for improved resolution. Signal amplification can be achieved through high-sensitivity chemiluminescent substrates (with femtogram detection limits) or fluorescent secondary antibodies with digital imaging systems for quantitative analysis. Extended primary antibody incubation (overnight at 4°C at 1:500 dilution) with gentle agitation improves sensitivity. For problematic samples, consider signal enhancement systems like biotin-streptavidin amplification or tyramide signal amplification, which can increase sensitivity by 10-100 fold. Finally, optimize transfer conditions by using PVDF membranes with 0.2 μm pore size and adding 0.1% SDS to the transfer buffer to facilitate transfer of hydrophobic domains while maintaining a moderate methanol concentration (10-15%) to improve binding to the membrane.

What approaches resolve contradictory data when At4g39290 antibody results conflict with transcriptomic findings?

Resolving discrepancies between At4g39290 antibody-based protein detection and transcriptomic data requires a systematic multidimensional approach. First, verify the reliability of both datasets independently . For transcriptomics, perform RT-qPCR validation with multiple reference genes and primer sets targeting different regions of the At4g39290 transcript. For protein data, test multiple antibodies targeting different epitopes of the At4g39290 protein if available, and validate using knockout/knockdown lines. Consider the following biological explanations for observed discrepancies: (1) post-transcriptional regulation through miRNAs or RNA-binding proteins affecting translation efficiency; (2) protein stability differences under experimental conditions; or (3) protein translocation between cellular compartments affecting extraction efficiency.

To investigate these possibilities, implement:

• Polysome profiling to assess translation efficiency of At4g39290 mRNA

• Pulse-chase experiments with protein synthesis inhibitors to measure protein half-life

• Proteasome inhibitor treatments to detect rapid protein turnover

• Comprehensive subcellular fractionation to identify potential localization changes

This integrated approach can identify the regulatory level responsible for the observed discrepancy and provide insight into the specific mechanisms controlling At4g39290 expression.

How can I perform co-immunoprecipitation with At4g39290 antibody to identify interaction partners?

Co-immunoprecipitation (Co-IP) with At4g39290 antibody requires careful optimization to maintain protein complex integrity while minimizing non-specific interactions. Begin with freshly harvested tissue (preferably 3-4 week old rosettes or stress-treated seedlings where At4g39290 expression is highest) and extract proteins using a gentle lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100, 1 mM EDTA, and complete protease inhibitor cocktail . Pre-clear lysates with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding. For the IP, conjugate 2-5 μg of At4g39290 antibody to magnetic Protein A/G beads using a chemical cross-linker like BS3 or DSS to prevent antibody co-elution and contamination of the final sample.

Incubate pre-cleared lysates with antibody-conjugated beads overnight at 4°C with gentle rotation. Implement stringent washing steps (at least 5 washes) using buffers with increasing salt concentrations (150-300 mM NaCl) to reduce non-specific interactions while maintaining specific complexes. For elution, use a gentle approach with competitive elution using the immunizing peptide (100-200 μg/ml) rather than harsh denaturing conditions, especially if maintaining native complexes is important. Analyze eluates using mass spectrometry with label-free quantification, comparing results to control IPs performed with non-specific IgG or using At4g39290 knockout tissue as negative controls. For validation of identified interactions, perform reverse Co-IPs using antibodies against the putative interacting partners, and confirm biological relevance through techniques like bimolecular fluorescence complementation or FRET analysis in planta.

How do different fixation methods affect At4g39290 antibody performance in immunohistochemistry?

The following table summarizes comparative effectiveness of different fixation methods for At4g39290 detection:

For optimal results with At4g39290 detection, a dual fixation approach using 2% paraformaldehyde followed by brief (30-second) post-fixation with methanol often provides the best combination of structural preservation and epitope accessibility. For tissues with high levels of phenolic compounds that may interfere with antibody binding, incorporating polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP) in the fixative solution can improve antibody penetration and reduce non-specific background.

What are the best strategies for quantifying At4g39290 protein levels across different developmental stages?

Quantifying At4g39290 protein levels across developmental stages requires an integrated approach that combines multiple quantitative techniques. Western blotting with fluorescent secondary antibodies provides the foundation for basic quantification, but must be rigorously controlled . Always normalize At4g39290 signals to multiple loading controls (not just a single housekeeping protein), preferably using stain-free gel technology or total protein normalization through Ponceau S staining to avoid biases from housekeeping gene regulation during development. For developmental series, analyze 3-5 biological replicates per stage with technical duplicates, and implement randomized gel loading patterns to control for position effects during transfer.

For more precise quantification, enzyme-linked immunosorbent assay (ELISA) calibrated with recombinant At4g39290 protein standards can provide absolute quantification in ng/mg total protein. Consider developing a sandwich ELISA using two antibodies targeting different epitopes of At4g39290 for enhanced specificity. For tissue-specific and subcellular resolution, combine immunohistochemistry with digital image analysis, using software like ImageJ with appropriate thresholding and background correction. To account for developmental changes in cellular composition, complement protein-level data with transcript analysis using stage-specific RNA-seq or RT-qPCR.

The following quantification workflow is recommended:

• Harvest tissue from multiple plants at precisely defined developmental stages (based on established markers like days after germination, leaf number, or reproductive stage)

• Process parallel samples for protein extraction (Western blot/ELISA) and fixation (immunohistochemistry)

• Quantify protein using at least two independent methods

• Normalize to appropriate reference points (total protein, cell number, tissue volume)

• Statistically analyze using mixed-effects models that account for biological and technical variability

This approach provides robust quantification across developmental stages while controlling for the numerous variables that can confound developmental studies.

How can I design experiments to determine if phosphorylation affects At4g39290 antibody recognition?

Designing experiments to assess phosphorylation effects on At4g39290 antibody recognition requires a systematic approach that distinguishes between genuine phosphorylation events and epitope masking. Begin by examining the antibody's epitope sequence for potential phosphorylation sites using phosphorylation prediction tools like PhosPhAt for plant-specific predictions . If the epitope contains predicted phosphorylation sites, design a multi-phase experimental approach:

First, generate parallel protein samples from the same tissue source with differential phosphorylation states:

• Untreated extract (native phosphorylation)

• Phosphatase-treated extract (dephosphorylated)

• ATP/kinase-treated extract (enhanced phosphorylation)

• Phosphatase inhibitor-treated extract (maximally phosphorylated)

Compare antibody recognition across these samples using Western blotting. A phosphorylation-sensitive antibody will show differential binding patterns, typically with reduced signal in the phosphorylated samples if the phosphorylation occurs within the epitope region.

For confirmation, utilize phospho-specific antibodies if available, or employ Phos-tag SDS-PAGE, which reduces the mobility of phosphorylated proteins. Run identical samples on standard and Phos-tag gels, and compare band patterns when probed with the At4g39290 antibody. Shifts in apparent molecular weight in the Phos-tag gel indicate phosphorylated forms.

To identify the specific phosphorylation sites affecting antibody recognition, perform immunoprecipitation of At4g39290 followed by phosphopeptide enrichment and mass spectrometry analysis. Then synthesize peptides corresponding to the epitope in both phosphorylated and non-phosphorylated forms, and test antibody binding using techniques like peptide ELISA or surface plasmon resonance. This comprehensive approach not only determines whether phosphorylation affects antibody recognition but also identifies the specific sites involved and quantifies the magnitude of the effect.

How can I address non-specific binding when using At4g39290 antibody in plant tissue with high phenolic content?

Non-specific binding of At4g39290 antibody in plant tissues with high phenolic content represents a significant challenge for immunological studies, particularly in mature or stress-treated Arabidopsis tissues. This issue results from phenolic compounds forming complexes with proteins and antibodies, leading to high background and false positives . A systematic approach to mitigate these issues involves modifying both sample preparation and immunodetection protocols.

For sample preparation, incorporate phenolic-neutralizing agents in your extraction/fixation buffers:

• Add 2-5% (w/v) polyvinylpyrrolidone (PVP-40) and 2% polyvinylpolypyrrolidone (PVPP) to extraction buffers

• Include 50-100 mM sodium metabisulfite as an antioxidant

• Supplement with 5-10 mM ascorbic acid and 5-10 mM dithiothreitol to prevent phenolic oxidation

• Extract at 4°C under dim lighting conditions to minimize oxidation reactions

For immunodetection protocols, modify blocking and antibody incubation steps:

• Extend blocking time to 2-3 hours using 5% non-fat dry milk with 1% BSA in TBS-T

• Add 0.1-0.3% Triton X-100 to blocking and antibody solutions to reduce hydrophobic interactions

• Include 20 mM glycine in washing buffers to quench residual aldehyde groups from fixation

• Incorporate 5% normal serum from the same species as the secondary antibody host

• Reduce primary antibody concentration and extend incubation time (use 1:1000 dilution overnight at 4°C)

For particularly problematic samples, consider tissue pre-treatment with gentle oxidizing agents like sodium metaperiodate (5-10 mM) to modify phenolic structures before antibody application. Additionally, implementing a two-step detection system using biotinylated secondary antibodies with streptavidin-conjugated reporter molecules can increase specificity by introducing an additional binding event. Always process comparable tissues from At4g39290 knockout plants as negative controls to establish the true level of non-specific binding under your specific experimental conditions.

What are the critical factors affecting reproducibility in At4g39290 antibody-based chromatin immunoprecipitation (ChIP) experiments?

Reproducibility in At4g39290 antibody-based ChIP experiments depends on optimizing several critical parameters that affect chromatin quality, antibody specificity, and downstream processing . The most fundamental consideration is confirming whether At4g39290 directly interacts with DNA or functions as part of a chromatin-associated complex, as this determines optimal crosslinking conditions.

For formaldehyde crosslinking, optimize both concentration (1-3%) and incubation time (5-15 minutes) for your specific tissue type. Over-crosslinking can mask epitopes and reduce immunoprecipitation efficiency, while under-crosslinking fails to capture transient interactions. For Arabidopsis seedlings, initial testing with 1% formaldehyde for 10 minutes often provides a good starting point, but systematically test variations to determine optimal conditions for At4g39290 detection.

Chromatin fragmentation represents another critical variable. Sonication parameters (amplitude, cycle number, duration) must be carefully optimized and standardized, aiming for fragments between 200-500 bp. Verify fragmentation efficiency via agarose gel electrophoresis before proceeding to immunoprecipitation. Using biological replicates processed on different days is essential for establishing reproducibility.

The following table highlights key variables and their optimization targets:

Finally, implement rigorous controls including IgG control, no-antibody control, and ideally ChIP in At4g39290 knockout/knockdown lines. For data analysis, normalize to multiple input references and employ appropriate statistical methods that account for the compositional nature of ChIP-seq data. Biological interpretation should integrate results with transcriptomics, DNA accessibility data, and known protein interaction networks to establish functional relevance of At4g39290 chromatin associations.

What is the significance of epitope selection when generating new At4g39290 antibodies for different experimental applications?

Epitope selection fundamentally determines antibody performance across different experimental applications when generating new At4g39290 antibodies . The optimal epitope varies significantly depending on the intended application, structural context of the protein, and experimental conditions. For At4g39290, which contains multiple functional domains, strategic epitope targeting enables application-specific antibody development.

For applications requiring native protein recognition (immunoprecipitation, ChIP), select epitopes from exposed regions like loops or terminal domains that remain accessible in the folded protein. Hydrophilic stretches with flexible secondary structure and minimal post-translational modification sites typically perform best. Conversely, for Western blot applications where proteins are denatured, internal sequences can serve as effective epitopes, even if normally buried in the native structure.

Computational epitope prediction combines several parameters for optimal selection:

For immunohistochemistry applications targeting fixed tissues, epitopes containing few lysine and arginine residues (which are primary targets for fixative crosslinking) often maintain better accessibility. For generating antibodies suitable for multiplexing (e.g., co-localization studies), coordinate epitope selection with existing antibodies to allow simultaneous detection without interference.

When developing antibodies for distinguishing between protein isoforms or family members, focus on regions with maximum sequence divergence. For At4g39290 specifically, target unique sequences not present in its closest homologs to ensure specificity. Finally, for quantitative applications like ELISA, prioritize epitopes with linear structure and consistent binding kinetics, preferably designing sandwich assays using antibodies targeting distant epitopes to maximize specificity and sensitivity.

How can CRISPR-generated At4g39290 variants be used to map epitope binding regions and validate antibody specificity?

CRISPR-Cas9 gene editing provides powerful tools for precise epitope mapping and antibody validation through strategic modification of the At4g39290 gene . By generating a series of targeted mutations within the suspected epitope region, researchers can systematically determine the critical residues required for antibody recognition. This approach offers superior resolution compared to traditional epitope mapping and validates antibody specificity in an endogenous context.

Begin by designing multiple guide RNAs targeting various regions of the At4g39290 coding sequence, prioritizing regions predicted to contain the epitope based on the immunizing peptide information. For comprehensive epitope mapping, create three categories of CRISPR-modified plants:

• Domain deletion lines: Remove entire structural domains (10-30 amino acids) to broadly localize the epitope

• Small deletion lines: Generate 3-6 amino acid deletions across the suspected epitope region

• Point mutation lines: Introduce single amino acid substitutions at critical positions, particularly changing charge or size properties

Following genotypic validation, analyze protein expression in these modified lines using the At4g39290 antibody via Western blotting. Complete loss of signal identifies regions essential for antibody binding. For lines with reduced but not eliminated binding, quantify the relative signal reduction to identify residues contributing partially to epitope recognition.

For more detailed analysis, combine CRISPR editing with heterologous expression systems:

• Clone wild-type and CRISPR-modified At4g39290 sequences into expression vectors

• Express in heterologous systems (E. coli, yeast, or in vitro translation)

• Perform dot blots or Western blots with serial dilutions to quantify binding affinity changes

• Use surface plasmon resonance to determine precise binding kinetics alterations

This integrated approach provides a complete map of the antibody epitope while simultaneously generating valuable resources for studying At4g39290 function. The resulting collection of epitope-modified plants serves dual purposes: validating antibody specificity and providing tools for structure-function analysis of the At4g39290 protein in vivo.

What are the most effective strategies for combining At4g39290 antibody detection with RNA-FISH for correlative protein-transcript analysis?

Correlative protein-transcript analysis combining At4g39290 antibody immunodetection with RNA fluorescence in situ hybridization (RNA-FISH) requires careful protocol optimization to preserve both protein epitopes and RNA integrity . The key challenge lies in developing a unified workflow that maintains both antibody binding capacity and RNA hybridization efficiency while avoiding spatial displacement of either signal.

Begin with a fixation protocol that adequately preserves both protein and RNA: 15-minute treatment with 4% paraformaldehyde in PBS supplemented with 0.1% glutaraldehyde and 0.1% Triton X-100 generally provides a good starting point. This mild crosslinking preserves tissue architecture while maintaining RNA accessibility. For Arabidopsis tissues, vacuum infiltration during fixation significantly improves reagent penetration.

For sequential detection, the order of procedures critically affects success rates. The optimal workflow typically follows this sequence:

• Perform immunodetection of At4g39290 first, using fluorophore-conjugated secondary antibodies with good photostability (Alexa Fluor series)

• Post-fix briefly (10 minutes) with 2% paraformaldehyde to stabilize antibody-antigen complexes

• Treat with 0.1% acetylated BSA and 0.1% RNase inhibitor to prevent non-specific RNA binding

• Apply pre-hybridization solution containing 50% formamide, 5× SSC, 50 μg/ml heparin, and 0.1% Tween-20

• Hybridize with fluorescently-labeled RNA probes targeting At4g39290 transcript (typically 5-6 probes of 50 nucleotides each for optimal sensitivity)

• Perform stringent washes with decreasing SSC concentrations

• Mount in anti-fade medium containing DAPI for nuclear counterstaining

For multiplexed detection, utilize spectrally distinct fluorophores (e.g., Alexa 488 for protein, Alexa 594 for RNA) and perform careful controls to ensure minimal spectral bleed-through. Additional considerations include using tyramide signal amplification for low-abundance targets and implementing computational deconvolution approaches to enhance signal clarity.

This correlative approach provides unique insights into the spatiotemporal relationship between At4g39290 transcript and protein localization, revealing potential post-transcriptional regulatory mechanisms and subcellular trafficking patterns that cannot be detected by either method alone.

How can proximity labeling methods be integrated with At4g39290 antibodies to identify transient protein interactions?

Integrating proximity labeling with At4g39290 antibodies creates a powerful system for capturing transient and weak protein interactions that traditional co-immunoprecipitation might miss . This approach is particularly valuable for studying At4g39290's dynamic interactions during stress responses or developmental transitions. Two primary proximity labeling strategies can be effectively combined with At4g39290 antibody-based studies:

The first approach utilizes antibody-enzyme fusion constructs where peroxidase (APEX2) or biotin ligase (TurboID/miniTurboID) is directly conjugated to purified At4g39290 antibodies. These conjugated antibodies are then introduced into permeabilized plant cells or applied to tissue sections, where they bind endogenous At4g39290 protein and label proximal proteins through either biotin-phenol oxidation (APEX2) or direct biotinylation (TurboID). This method preserves the native cellular context and requires no genetic modification but depends heavily on antibody specificity and tissue permeability.

The second, more common approach involves generating transgenic plants expressing At4g39290 fused to an enzymatic proximity labeling tag:

• Create C-terminal and N-terminal TurboID or APEX2 fusions with At4g39290 under native promoter control

• Validate fusion protein functionality and localization using the At4g39290 antibody

• Perform in vivo proximity labeling by supplying the appropriate substrate (biotin-phenol for APEX2 or biotin for TurboID)

• Extract and purify biotinylated proteins using streptavidin beads

• Identify interacting partners through mass spectrometry

• Validate key interactions with reverse labeling and co-localization studies

For temporal resolution of interaction dynamics, implement a time-course analysis with short labeling windows (5-10 minutes for APEX2, 10-30 minutes for TurboID) across different conditions or developmental stages. Statistical analysis of interaction profiles can reveal condition-specific partners and interaction networks.

The following table compares key parameters for proximity labeling methods applicable to At4g39290 interaction studies:

This integrated approach provides a dynamic view of At4g39290 interaction networks that complements traditional protein-protein interaction methods, particularly for capturing weak or transient interactions that may be functionally significant in plant stress responses.