At4g24830 Antibody

What is At4g24830 and why is it studied in Arabidopsis research?

At4g24830 is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress), identified by its UniProt accession number Q9SZX3. The protein is studied primarily in plant molecular biology research focusing on Arabidopsis as a model organism. Antibodies against this protein are valuable tools for tracking its expression and localization. Research with At4g24830 antibodies enables investigation of protein function within various plant tissues and under different environmental conditions, contributing to our understanding of plant molecular mechanisms .

What applications are suitable for At4g24830 antibody?

At4g24830 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications. These techniques allow researchers to detect and quantify At4g24830 protein in plant tissue samples. While not explicitly validated for immunohistochemistry or immunofluorescence, researchers might adapt protocols from similar plant antibodies like the Anti-Rubisco activase antibody, which has been successfully used across multiple plant species including Arabidopsis thaliana .

What are the optimal storage conditions for At4g24830 antibody?

For maximum stability and activity retention, At4g24830 antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles significantly reduce antibody performance, so aliquoting the antibody upon first thaw is strongly recommended. For short-term storage (1-2 weeks), 4°C is acceptable, but prolonged storage should be at -20°C or below. The antibody is typically stored in buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as preservative .

How should I prepare plant samples for At4g24830 antibody detection?

Sample preparation for At4g24830 detection requires careful extraction to preserve protein integrity. Based on protocols for similar plant antibodies, I recommend:

• Grind 100-200 mg of fresh or frozen plant tissue in liquid nitrogen

• Add extraction buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail)

• Homogenize and centrifuge at 12,000g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration

• For Western blot applications, denature samples with SDS loading buffer at 95°C for 5 minutes

• Load 10-20 μg of protein per lane for detection

How can I optimize Western blot conditions for At4g24830 antibody?

Optimization of Western blot conditions for At4g24830 antibody requires systematic adjustment of multiple parameters. Based on protocols for similar plant antibodies:

Critically, sample denaturation conditions significantly impact detection. For optimal results with plant proteins similar to At4g24830, a buffer containing 0.1M Na₂CO₃ and 0.1M DTT with 2% SDS-5% sucrose, heating at 100°C for 1 minute has been effective .

How can I assess antibody specificity for At4g24830 detection?

Rigorous validation of At4g24830 antibody specificity is essential for reliable research outcomes. I recommend a multi-step validation approach:

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide (when available) before applying to Western blot or ELISA. Disappearance of signal confirms specificity.

• Knockout/knockdown controls: Use CRISPR/Cas9 or RNAi lines with reduced or eliminated At4g24830 expression. Compare signal intensity between wild-type and modified plants.

• Heterologous expression: Express recombinant At4g24830 protein in a system like E. coli and detect with the antibody.

• Mass spectrometry validation: Immunoprecipitate the protein using the antibody and confirm identity through mass spectrometry.

• Cross-reactivity testing: Test the antibody against related plant species to identify potential cross-reactivity, similar to testing performed with antibodies like Anti-Rubisco activase .

What endogenous controls should be used with At4g24830 antibody in expression studies?

Selection of appropriate endogenous controls is crucial for normalizing At4g24830 expression data. Based on Arabidopsis research protocols:

When studying stress responses or developmental changes, multiple controls should be analyzed to ensure consistent normalization across experimental conditions .

How can I address weak or absent signal when using At4g24830 antibody?

Weak or absent signals when using At4g24830 antibody may stem from several factors. A systematic troubleshooting approach should include:

• Extraction optimization: Ensure complete protein extraction by testing different buffer compositions. For Arabidopsis proteins, adding 0.1M DTT and/or 2% SDS can improve extraction.

• Protein denaturation: Some plant proteins require specific denaturation conditions. Try varying temperature (65°C, 85°C, 100°C) and duration (1-10 minutes).

• Enrichment techniques: Consider enriching for the protein of interest through fractionation or immunoprecipitation before detection.

• Epitope masking: Post-translational modifications may mask epitopes. Treatment with phosphatases or deglycosylation enzymes may improve detection.

• Antibody concentration: Increase antibody concentration gradually up to 1:500 if signal remains weak.

• Enhanced detection systems: Switch to more sensitive detection methods like enhanced chemiluminescence (ECL) plus or super signal systems .

What strategies can address non-specific binding when using At4g24830 antibody?

Non-specific binding is a common challenge when working with plant antibodies like At4g24830. Evidence-based approaches to improve specificity include:

• Blocking optimization: Test different blocking agents (milk, BSA, casein, commercial blockers) at varying concentrations (3-7%) and durations (1-4 hours).

• Antibody dilution series: Perform a dilution series (1:1,000 to 1:10,000) to identify optimal concentration balancing specific signal and background.

• Stringent washing: Increase wash buffer stringency with higher detergent concentrations (0.1-0.3% Tween-20) or salt concentrations (150-500 mM NaCl).

• Pre-adsorption: Pre-adsorb antibody with plant extract from species with low or no target protein expression.

• Alternative buffers: For particularly problematic samples, RIPA buffer (with 0.1% SDS, 0.5% sodium deoxycholate) may reduce non-specific interactions .

How does antibody structure influence At4g24830 detection specificity?

Antibody structure fundamentally impacts At4g24830 detection specificity through the organization of complementarity-determining regions (CDRs). Research on antibody-antigen interactions reveals:

The polyclonal nature of At4g24830 antibody means it contains multiple antibody clones recognizing different epitopes on the target protein. CDRs, particularly CDR-H3 (heavy chain CDR3), show high length diversity and amino acid variability, with increased usage of aromatic residues (especially tyrosine), charged and polar residues (aspartic acid, serine), and the flexible residue glycine .

Specific residue positions within each CDR influence antigen recognition, with a unique amino acid distribution pattern. Compared to non-CDR regions, CDRs show increased frequency of tyrosine, serine, aspartic acid, and arginine. These structural features determine the specificity and affinity of the antibody for At4g24830 protein .

For researchers developing improved At4g24830 detection methods, understanding these structural considerations can guide optimization efforts, particularly when troubleshooting cross-reactivity issues or designing new detection reagents.

How can At4g24830 antibody be used to investigate protein-protein interactions?

At4g24830 antibody can be employed in multiple sophisticated approaches to study protein-protein interactions:

• Co-immunoprecipitation (Co-IP): The antibody can capture At4g24830 protein along with its interaction partners from plant extracts. This requires:

  • Gentle lysis conditions (150 mM NaCl, 1% NP-40 or Triton X-100, 50 mM Tris pH 7.5)

  • Incubation with antibody (typically 2-5 μg per mg of protein lysate)

  • Capture with Protein A/G beads

  • Analysis of precipitated complexes by Western blot or mass spectrometry

• Proximity ligation assay (PLA): For detecting in situ protein interactions with spatial resolution, combining At4g24830 antibody with antibodies against suspected interaction partners.

• Chromatin immunoprecipitation (ChIP): If At4g24830 has DNA-binding properties or associates with chromatin-bound proteins, ChIP can identify genomic binding sites.

• Bimolecular fluorescence complementation (BiFC): While not directly using the antibody, BiFC complements antibody-based approaches by visualizing interactions in living cells .

What considerations are important when using At4g24830 antibody in developmental studies?

Developmental studies using At4g24830 antibody require careful experimental design to account for temporal and spatial protein expression patterns:

• Tissue-specific extraction protocols: Different plant tissues require modified extraction procedures. Seed tissues, for example, require:

  • Specific buffer components (0.1M Na₂CO₃, 0.1M DTT)

  • Higher detergent concentrations (2% SDS)

  • Different homogenization techniques than leaf tissue

• Developmental timing: At4g24830 expression may vary significantly across developmental stages. For seed development studies, careful staging is essential, with protein accumulation often increasing dramatically between 8-14 days post-anthesis (dpa) .

• Whole-plant analysis: For comprehensive developmental studies, examine multiple tissues at different stages, potentially creating a developmental expression atlas.

• Protein stability considerations: Post-translational modifications or protein degradation patterns may change during development, affecting antibody detection efficiency.

• Transgenic approaches: Consider complementing antibody studies with promoter-reporter fusions (GUS, GFP) to track expression patterns at cellular resolution .

How can At4g24830 antibody be used in stress response studies?

At4g24830 antibody can be deployed strategically in plant stress response studies to monitor protein-level changes that may not be evident at the transcriptional level:

• Stress-specific extraction protocols: Different stress treatments may necessitate modified extraction protocols to account for changes in cellular composition. For example, salt stress often requires higher salt concentrations in extraction buffers.

• Temporal resolution sampling: Create a detailed time-course experiment with samples collected at multiple time points (0, 1, 3, 6, 12, 24, 48 hours) after stress application to capture dynamic protein responses.

• Subcellular fractionation: Combine with cellular fractionation techniques to determine if stress causes relocalization of At4g24830 protein between cellular compartments.

• Post-translational modification analysis: Use phospho-specific antibodies or mass spectrometry in conjunction with At4g24830 antibody to detect stress-induced modifications.

• Comparative stress analysis: Apply consistent protocols across different stress treatments (drought, salt, heat, cold, pathogen) to identify stress-specific versus general stress responses .

What considerations are important when integrating At4g24830 antibody data with transcriptomic analyses?

Integrating protein-level data from At4g24830 antibody studies with transcriptomic data requires addressing several methodological challenges:

• Temporal dynamics: Transcript and protein levels often show time-shifted relationships. Design experiments with staggered sampling to capture both transcript accumulation and subsequent protein synthesis.

• Sample preparation harmonization: Extract RNA and protein from the same tissue samples when possible to minimize biological variability.

• Quantification methods: For precise correlation:

  • Use quantitative Western blotting with standard curves for protein quantification

  • Normalize protein levels against consistent loading controls

  • Compare with normalized transcript counts from RNA-seq or qPCR data

• Statistical integration approaches: Apply appropriate statistical methods for integrating datasets with different dynamic ranges and error distributions.

• Validation experiments: Design targeted experiments to test hypotheses about discrepancies between transcript and protein levels, including investigations of:

  • Post-transcriptional regulation

  • Protein stability differences

  • Translational efficiency

How can recombinant antibody production systems in plants affect At4g24830 antibody quality?

The plant-based recombinant antibody production systems can significantly impact At4g24830 antibody quality through several mechanisms:

Research shows that seed-specific recombinant antibody production in Arabidopsis triggers endoplasmic reticulum stress and unfolded protein response (UPR). Microarray analysis of antibody-expressing lines revealed significant differential expression of numerous genes compared to wild-type plants. Specifically, 73-84 genes were up-regulated across different antibody-expressing lines, with 27 commonly up-regulated genes across all lines .

This cellular stress response can affect:

• Glycosylation patterns: Plant-specific glycosylation differs from mammalian patterns, potentially affecting antibody function.

• Folding efficiency: UPR activation indicates folding challenges, which may reduce the percentage of correctly folded, functional antibody molecules.

• Yield variation: Different antibody formats accumulated at varying levels (from 0.05% to 2.64% of total soluble protein) at different developmental stages.

• Batch consistency: The stress response may vary between production batches, affecting consistency.

For researchers using At4g24830 antibody, understanding these production-related variables can help interpret inconsistencies between antibody batches and guide selection of optimal detection conditions .