GH3.4 Antibody

What is GH3.4 and what is its biological significance?

GH3.4 is a member of the GH3 (Gretchen Hagen 3) protein family found in plants, particularly well-studied in Oryza sativa subsp. japonica (Rice). GH3 proteins generally function as acyl acid amido synthetases that conjugate plant hormones to amino acids, playing critical roles in hormone homeostasis and plant development. The GH3.4 variant specifically has been identified with UniProt Number Q60EJ6 and Entrez Gene ID 4339234 . These proteins are particularly important in auxin and jasmonate signaling pathways, contributing to plant growth regulation, development, and stress responses.

How are GH3.4 antibodies generated for research use?

Commercial GH3.4 antibodies, such as those available from suppliers like Cusabio, are typically generated using recombinant Oryza sativa subsp. japonica GH3.4 protein as the immunogen . The production process involves immunizing rabbits with the purified recombinant protein, followed by affinity purification of the resulting polyclonal antibodies using epitope-specific immunogen chromatography . This process yields high-specificity antibodies that recognize the native GH3.4 protein in plant tissues, particularly rice.

What are the primary applications for GH3.4 antibodies?

Based on available documentation, GH3.4 antibodies are primarily validated for Western Blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . These techniques allow researchers to detect and quantify GH3.4 protein expression in plant tissues. The antibodies are delivered with positive control antigens (200μg) and pre-immune serum (1ml) as a negative control to facilitate experimental validation . While immunohistochemistry applications might be possible, they would require additional optimization and validation by individual researchers.

How should researchers optimize Western Blot protocols for GH3.4 detection?

For optimal Western Blot performance with GH3.4 antibodies, researchers should consider the following protocol adjustments:

• Sample preparation: Use a buffer containing protease inhibitors to prevent degradation of the GH3.4 protein during extraction from plant tissues.

• Dilution optimization: Begin with the manufacturer's recommended dilution range of 1:500-2000 and optimize based on signal-to-noise ratio in your specific samples.

• Blocking strategy: Use a 5% BSA solution rather than milk-based blockers, as the latter may interfere with plant protein detection.

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C to enhance specific binding, especially when working with lower abundance proteins in plant extracts.

• Controls: Always include the provided positive control antigens and pre-immune serum as controls .

The ability to detect GH3.4 in Western Blots makes this technique valuable for studying protein expression changes under different experimental conditions, such as hormone treatments or environmental stresses.

What cross-reactivity considerations should researchers account for?

While the GH3.4 antibody is developed against rice (Oryza sativa) GH3.4, researchers working with other plant species should perform careful validation. GH3 proteins share conserved domains across species, potentially allowing cross-reactivity, but sequence variations may affect antibody recognition. Preliminary testing with known positive and negative controls is essential when extending use to non-validated species. When cross-reactivity is observed, researchers should confirm specificity through additional techniques such as immunoprecipitation followed by mass spectrometry or parallel gene expression analysis.

How does sample preparation affect GH3.4 antibody performance?

Effective sample preparation is critical for successful GH3.4 detection. Consider the following methodological approaches:

• Tissue selection: Target tissues known to express GH3.4, such as developing seeds, young leaves, or hormone-treated tissues.

• Extraction buffer optimization: Use buffers containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and a comprehensive protease inhibitor cocktail.

• Protein preservation: Flash-freeze tissues in liquid nitrogen before processing to preserve protein integrity.

• Subcellular fractionation: Consider subcellular fractionation protocols if interested in localization of GH3.4 protein.

• Denaturing conditions: Optimize denaturing conditions during sample preparation, as plant proteins often require stronger denaturation due to complex matrices.

What are common issues encountered with GH3.4 antibodies and how can they be addressed?

When working with GH3.4 antibodies, researchers might encounter several challenges:

• High background: If experiencing high background in Western blots, increase the number and duration of washing steps, optimize blocking conditions, and consider using the pre-immune serum provided with the antibody as a negative control .

• Weak or absent signal: For weak signals, consider:

  • Increasing protein loading (up to 50-80μg total protein)

  • Reducing antibody dilution (try 1:500)

  • Extending exposure time or using more sensitive detection methods

  • Confirming GH3.4 expression in your specific tissues/conditions

• Multiple bands: Multiple bands may represent:

  • Post-translational modifications of GH3.4

  • Cross-reactivity with other GH3 family members

  • Protein degradation products

  Use the provided positive control antigen to identify the correct band size .

How do detection methods compare for GH3.4 antibody applications?

Different detection methods offer varying advantages for GH3.4 research:

The choice of detection method should align with experimental goals and available equipment.

How should researchers interpret contradictory results between GH3.4 antibody detection and gene expression data?

Discrepancies between protein detection (antibody-based) and mRNA expression (RT-PCR, RNA-seq) are common and biologically meaningful. When confronted with contradictory results:

• Confirm antibody specificity: Validate antibody specificity using knockout/knockdown lines if available.

• Consider post-transcriptional regulation: Many proteins show temporal delays between transcription and translation, or undergo regulation at the translational level.

• Assess protein stability: GH3.4 protein may have different turnover rates than its mRNA.

• Examine experimental timing: Sample collection timing may capture different stages of the response.

• Perform time-course experiments: Conducting both protein and mRNA analyses across a time-course can reveal the temporal relationship between transcription and translation.

The most comprehensive understanding comes from integrating both protein-level (antibody-based) and transcript-level data.

How do GH3.4 antibodies compare to antibodies against other GH3 family members?

The GH3 protein family includes multiple members with distinct but related functions. When comparing GH3.4 antibodies to those targeting other GH3 family proteins:

• Specificity considerations: Antibodies raised against specific GH3 members may cross-react with other family members due to conserved domains. Epitope selection during antibody generation is critical for specificity.

• Application breadth: While the GH3.4 antibody from Cusabio is validated for ELISA and Western Blot applications , antibodies against other GH3 members may have different validation profiles, including immunohistochemistry or immunofluorescence capabilities.

• Species reactivity: GH3.4 antibodies are primarily validated for plant species detection , while some antibodies against GH3 domain-containing proteins in animals may have different reactivity profiles, including human and mouse as seen with the GHDC antibody .

What methodological approaches can enhance GH3.4 detection in difficult tissues?

Some plant tissues present challenges for protein extraction and antibody detection. Consider these methodological enhancements:

• Tissue-specific extraction buffers: For recalcitrant tissues like seeds or roots, modify standard extraction buffers with increased detergent concentrations (2-3% Triton X-100) or include chaotropic agents.

• Protein concentration techniques: Implement protein precipitation methods (TCA/acetone or methanol/chloroform) to concentrate GH3.4 from dilute extracts.

• Epitope retrieval techniques: For fixed tissues in immunohistochemistry, heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) may improve antibody accessibility.

• Signal amplification systems: Consider biotin-streptavidin amplification or tyramide signal amplification for very low abundance detection.

• Membrane optimization: PVDF membranes often provide better protein retention than nitrocellulose for plant proteins, improving detection sensitivity.

How can GH3.4 antibodies be used to study hormone signaling pathways?

GH3.4 antibodies provide valuable tools for investigating hormone signaling networks:

• Hormone-induced expression changes: Monitor GH3.4 protein levels in response to auxin, jasmonate, or other hormonal treatments using Western blot or ELISA, comparing results with appropriate controls.

• Co-immunoprecipitation studies: Use GH3.4 antibodies to identify protein interaction partners in hormone signaling pathways, providing insights into functional complexes.

• Subcellular localization: Determine if GH3.4 protein localization changes under different hormonal treatments using cellular fractionation followed by Western blot analysis.

• Protein stability analysis: Investigate GH3.4 protein turnover rates in response to hormone signaling using cycloheximide chase experiments with antibody detection.

• Comparative analysis across conditions: Create experimental designs that compare GH3.4 protein levels across different stress conditions, developmental stages, or genetic backgrounds.

How can GH3.4 antibodies contribute to stress response research in crops?

GH3.4 and related proteins play important roles in plant stress responses. Antibody-based approaches offer several advantages for studying these mechanisms:

• Protein-level stress markers: GH3.4 antibodies can be used to monitor protein-level changes in response to drought, salinity, temperature stress, or pathogen infection.

• Cultivar comparison: Comparing GH3.4 protein levels across stress-tolerant and susceptible cultivars may reveal correlations with adaptive responses.

• Temporal dynamics: Antibody detection can reveal the timing of GH3.4 protein accumulation during stress responses, which may differ from transcriptional dynamics.

• Post-translational modifications: With appropriate controls, antibodies might detect stress-induced modifications of GH3.4 through mobility shifts in Western blots.

• Stress signaling networks: Combine GH3.4 antibody detection with analyses of other stress-response proteins to build comprehensive signaling network models.

What quality control measures are essential for reproducible GH3.4 antibody research?

To ensure reproducible results with GH3.4 antibodies, implement these quality control measures:

• Antibody validation: Confirm specificity using positive control antigens provided by the manufacturer and test for cross-reactivity with related proteins.

• Lot-to-lot consistency: Maintain records of antibody lot numbers and perform validation experiments when switching lots.

• Standardized protocols: Develop and strictly adhere to standardized protocols for sample preparation, antibody dilution, incubation times, and washing procedures.

• Quantification standards: Include known quantity standards when performing quantitative analyses.

• Appropriate controls: Always include negative controls (pre-immune serum) and positive controls in each experiment.

• Storage conditions: Maintain antibodies according to manufacturer recommendations (-20°C or -80°C) and avoid repeated freeze-thaw cycles.

• Detailed documentation: Record all experimental conditions, including buffer compositions, incubation times, and detection methods.