At5g39365 Antibody

What is At5g39365 and why is it significant for plant research?

At5g39365 encodes a putative defensin-like protein 40 (DEFL family protein) in Arabidopsis thaliana, also known as mouse-ear cress . Defensin-like proteins typically function in plant immune responses and may have antimicrobial properties. The At5g39365 protein belongs to a large family of small cysteine-rich proteins that are widely distributed throughout the plant kingdom. Understanding this protein's function contributes to our knowledge of plant defense mechanisms and potentially to the development of disease-resistant crops.

What types of At5g39365 antibodies are available for research?

Based on available resources, researchers can access polyclonal antibodies against At5g39365, specifically rabbit anti-Arabidopsis thaliana At5g39365 polyclonal antibodies . These antibodies are typically antigen-affinity purified and belong to the IgG isotype. They are suitable for various applications including ELISA and Western blotting . The antibodies target specific epitopes of the Putative defensin-like protein 40, enabling researchers to study its expression, localization, and potential interactions.

What experimental applications are supported by At5g39365 antibodies?

At5g39365 antibodies have been validated for specific experimental applications including:

• Western blotting (WB) for protein expression analysis

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

• Potential applications in immunocytochemistry (though specific validation may be required)

These applications enable researchers to investigate protein expression patterns, tissue distribution, and potential roles in plant developmental processes or stress responses.

How should At5g39365 antibodies be validated before experimental use?

Proper validation of At5g39365 antibodies is crucial for experimental success and result reliability. A comprehensive validation approach should include:

• Specificity testing: Verify antibody recognition of the target protein using both recombinant At5g39365 protein and Arabidopsis tissue extracts

• Knockout/knockdown controls: If available, test the antibody against At5g39365 knockout/knockdown lines to confirm specificity

• Cross-reactivity assessment: Test against related DEFL family proteins to determine potential cross-reactivity

• Western blot validation: Confirm detection of a band at the expected molecular weight (approximately the predicted mass of the At5g39365 protein)

• Signal-to-noise ratio evaluation: Optimize antibody concentration to maximize specific signal while minimizing background

Experience from broader Arabidopsis antibody projects indicates that affinity purification significantly improves detection rates and specificity, with studies showing a marked improvement in signal quality after purification .

What are optimal protocols for Western blotting with At5g39365 antibodies?

For optimal Western blotting results with At5g39365 antibodies, consider the following methodological recommendations:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing protease inhibitors

  • Consider enrichment techniques for low-abundance proteins

  • Denature samples at 95°C for 5 minutes in SDS-PAGE loading buffer

• Gel electrophoresis and transfer:

  • Use 12-15% SDS-PAGE gels appropriate for lower molecular weight proteins

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

  • Verify transfer efficiency with reversible protein stains

• Antibody incubation:

  • Block with 3-5% BSA or non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary At5g39365 antibody (typically 1:1000 to 1:5000 dilution)

  • Wash thoroughly with TBST (at least 3 × 10 minutes)

  • Incubate with HRP-conjugated secondary antibody against rabbit IgG

• Detection:

  • Use ECL substrate for visualization

  • Expected band size should correspond to the predicted molecular weight of At5g39365

Experience from Arabidopsis antibody resources suggests that optimization of antibody concentration is critical, as the detection success rate has been shown to be approximately 55% for well-developed antibodies .

How can researchers verify the specificity of At5g39365 antibody signals?

Verifying antibody specificity is essential for confident interpretation of experimental results. For At5g39365 antibodies, consider these approaches:

• Peptide competition assay: Pre-incubate the antibody with excess purified At5g39365 protein or immunizing peptide before application to the sample. Disappearance of the signal confirms specificity.

• Multiple antibody validation: If possible, use antibodies raised against different epitopes of At5g39365 to confirm the same localization or expression pattern.

• Correlation with transcript levels: Compare protein detection with RT-qPCR data for At5g39365 expression across tissues or conditions.

• Genetic controls: Use T-DNA insertion lines, CRISPR knockouts, or RNAi lines with reduced At5g39365 expression to confirm signal reduction/elimination.

• Heterologous expression: Express tagged versions of At5g39365 in Arabidopsis or heterologous systems and confirm co-localization of antibody signal with the tag.

How can At5g39365 antibodies be utilized in subcellular localization studies?

Determining the subcellular localization of At5g39365 provides crucial insights into its function. Consider these methodological approaches:

• Immunofluorescence microscopy:

  • Fix Arabidopsis tissues with paraformaldehyde (typically 4%)

  • Perform cell wall digestion for better antibody penetration

  • Permeabilize with Triton X-100 or similar detergents

  • Block with BSA or serum to reduce non-specific binding

  • Incubate with At5g39365 antibody followed by fluorophore-conjugated secondary antibody

  • Co-stain with established organelle markers to determine precise localization

• Immunogold electron microscopy:

  • For higher resolution localization studies

  • Requires specialized sample preparation and ultra-thin sectioning

  • Incubate with At5g39365 antibody followed by gold-conjugated secondary antibody

  • Allows precise determination of protein localization at the ultrastructural level

• Biochemical fractionation:

  • Isolate subcellular fractions (membrane, cytosolic, nuclear, etc.)

  • Perform Western blotting on each fraction using At5g39365 antibody

  • Compare distribution pattern with known subcellular markers

When interpreting results, consider that defensin-like proteins may localize to different compartments depending on their stage in the secretory pathway or under different stress conditions.

What approaches can be used to study At5g39365 protein-protein interactions?

Understanding protein interaction networks provides valuable insights into At5g39365 function. Consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse Arabidopsis tissues under non-denaturing conditions

  • Immunoprecipitate using At5g39365 antibody

  • Analyze precipitated complexes by mass spectrometry or Western blot

  • Verify interactions by reciprocal Co-IP with antibodies against candidate interactors

• Proximity labeling coupled with immunoprecipitation:

  • Express BioID or TurboID fusions of At5g39365 in Arabidopsis

  • Allow biotinylation of proximal proteins

  • Purify biotinylated proteins and confirm presence of At5g39365 using its antibody

  • Identify interaction partners by mass spectrometry

• In situ proximity ligation assay (PLA):

  • Use At5g39365 antibody together with antibodies against candidate interactors

  • PLA signal occurs only when proteins are in close proximity (<40 nm)

  • Provides spatial information about interaction sites within cells

When designing these experiments, consider including appropriate controls and validating interactions through multiple independent methods.

How might sequence-based antibody design principles be applied to improve At5g39365 antibody performance?

Recent advances in antibody engineering can be applied to optimize At5g39365 antibodies:

As demonstrated in recent research, these approaches can yield antibodies with significantly improved binding affinity, potentially enhancing sensitivity in low-abundance protein detection scenarios .

What are appropriate experimental controls for At5g39365 antibody studies?

Robust experimental design requires appropriate controls:

How can researchers troubleshoot weak or absent At5g39365 antibody signals?

When facing challenges with At5g39365 detection, consider these methodological remedies:

• Protein extraction optimization:

  • Try different extraction buffers (varying detergents, salt concentrations)

  • Include protease inhibitors to prevent degradation

  • Consider denaturing vs. native conditions based on experimental goals

• Antibody concentration optimization:

  • Perform titration experiments to determine optimal concentration

  • Try longer incubation times (overnight at 4°C vs. 1-2 hours at room temperature)

• Signal enhancement strategies:

  • Use high-sensitivity detection systems (enhanced chemiluminescence)

  • Consider amplification systems (tyramide signal amplification for immunohistochemistry)

  • Try different blocking agents to reduce background while preserving specific signal

• Sample enrichment:

  • Consider immunoprecipitation prior to Western blotting for low-abundance proteins

  • Use tissue or cellular fractionation to concentrate the target protein

• Protein denaturation assessment:

  • If the antibody recognizes a conformational epitope, adjust denaturation conditions

  • Try different fixation protocols for immunohistochemistry

Experience from large-scale Arabidopsis antibody projects indicates that affinity purification can dramatically improve detection rates, with studies showing significant improvement after purification .

What experimental considerations are important when studying At5g39365 expression during stress responses?

When investigating At5g39365 regulation under stress conditions:

• Time-course design:

  • Include multiple time points to capture early, intermediate, and late responses

  • Consider both acute and chronic stress exposures

  • Sample at consistent times of day to control for circadian effects

• Tissue-specific analysis:

  • Different tissues may show distinct expression patterns

  • Consider microdissection for precise spatial resolution

  • Compare roots, shoots, and reproductive tissues

• Stress-specific protocols:

  • For biotic stress: Consider pathogen type, concentration, and inoculation method

  • For abiotic stress: Control intensity, duration, and recovery periods

  • For combined stresses: Apply systematic factorial designs

• Quantification approaches:

  • Use image analysis software for immunohistochemistry quantification

  • Apply densitometry for Western blot quantification

  • Include standard curves when possible for absolute quantification

• Data normalization:

  • Normalize to appropriate reference proteins that remain stable under the stress conditions

  • Consider multiple normalization strategies and compare results

How can single-cell approaches be integrated with At5g39365 antibody studies?

Emerging single-cell technologies offer new opportunities for At5g39365 research:

• Single-cell proteomics:

  • Adapt mass cytometry (CyTOF) protocols using metal-conjugated At5g39365 antibodies

  • Develop microfluidic-based single-cell Western blotting approaches

  • Investigate cell-to-cell variability in At5g39365 expression

• Spatial proteomics:

  • Apply multiplexed immunofluorescence with At5g39365 antibodies and other markers

  • Utilize imaging mass cytometry for spatial resolution of protein expression

  • Correlate with single-cell transcriptomics data for integrated analysis

• In situ protein quantification:

  • Develop quantitative immunofluorescence protocols for absolute protein quantification

  • Apply proximity ligation assays for protein complex quantification at single-cell resolution

These approaches could reveal heterogeneity in At5g39365 expression and function that would be masked in bulk tissue analyses.

What considerations are important when designing At5g39365 antibodies for multiplexed applications?

For simultaneous detection of multiple proteins including At5g39365:

• Antibody species selection:

  • Choose At5g39365 antibodies from different host species than other target antibodies

  • Consider isotype differences if antibodies are from the same species

• Signal separation strategies:

  • Select non-overlapping fluorophores for immunofluorescence

  • Use sequential blotting with stripping for Western blot applications

  • Consider chromogenic multiplex approaches with different substrates

• Cross-reactivity testing:

  • Perform extensive validation to ensure antibodies don't cross-react

  • Include appropriate controls in multiplexed experiments

  • Consider alternative epitopes if cross-reactivity occurs

Recent advances in antibody engineering, as demonstrated in the DyAb system, could potentially be applied to develop highly specific antibody variants with minimal cross-reactivity for multiplexed applications .