At2g34135 Antibody

What is At2g34135 and why is it significant for plant research?

At2g34135 is a gene in Arabidopsis thaliana that encodes a putative defensin-like protein 53. Defensin-like proteins play crucial roles in plant innate immunity against pathogens, acting as antimicrobial peptides that can disrupt microbial cell membranes. These proteins are increasingly important in understanding plant defense mechanisms, particularly as models for developing disease-resistant crops. The At2g34135 antibody enables detection and characterization of this protein, allowing researchers to study its expression patterns, subcellular localization, and potential functions in stress responses. When studying plant immunity, defensin-like proteins are often examined alongside other immunity-related proteins to establish comprehensive defense pathway models .

How can I confirm the specificity of my At2g34135 antibody?

Confirming antibody specificity requires multiple validation approaches. Start with Western blot analysis using both wild-type Arabidopsis tissue extracts and At2g34135 knockout/knockdown lines as controls. A specific antibody will show reduced or absent signal in knockout lines. Additionally, perform pre-absorption tests by incubating the antibody with purified recombinant At2g34135 protein prior to immunodetection. For definitive validation, immunoprecipitation followed by mass spectrometry can identify all proteins captured by the antibody. Finally, immunohistochemistry comparing wild-type and knockout plants can provide spatial validation of specificity. Document all validation results systematically, as antibody specificity can vary across experimental conditions and samples .

What is the recommended sample preparation protocol for optimal At2g34135 detection?

For optimal detection of At2g34135, careful sample preparation is essential. For plant tissue samples, use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail. Fresh tissue yields better results than frozen samples. Homogenize tissues thoroughly at 4°C and centrifuge at 14,000 × g for 15 minutes. For Western blot applications, avoid heat denaturation above 70°C as defensin-like proteins may aggregate. For immunoprecipitation, a gentler lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA with protease inhibitors) is recommended. For immunohistochemistry, fixation in 4% paraformaldehyde for 12-16 hours followed by embedding in paraffin or resin yields consistent results .

What are the validated applications for At2g34135 antibody?

The At2g34135 antibody has been validated for several experimental applications with specific optimization parameters for each. The primary validated applications include:

For optimal results in Western blotting, reducing conditions are recommended with 5% BSA as a blocking agent rather than milk products. In ELISA applications, overnight coating at 4°C with purified antibody yields more consistent results than shorter incubation periods. For immunohistochemistry, antigen retrieval using citrate buffer (pH 6.0) significantly improves detection sensitivity .

How should I optimize Western blotting conditions for At2g34135 antibody?

Optimizing Western blotting conditions for At2g34135 antibody requires careful attention to several parameters. First, sample preparation is critical—use fresh tissue when possible and extract in Tris buffer with 0.5% Triton X-100. Load 15-20 μg of total protein per lane and separate using a 12-15% SDS-PAGE gel to properly resolve the defensin-like protein. Transfer to PVDF membrane (rather than nitrocellulose) at 30V overnight at 4°C for optimal protein retention. For blocking, use 5% BSA in TBST for 2 hours at room temperature rather than milk products, which can interfere with antibody binding to plant proteins.

Primary antibody incubation should be performed at 1 μg/mL concentration overnight at 4°C with gentle agitation. After washing with TBST (5 washes, 5 minutes each), incubate with HRP-conjugated secondary antibody at 1:10,000 dilution for 90 minutes at room temperature. For development, enhanced chemiluminescent substrate with 15-30 second exposure typically yields optimal signal-to-noise ratio. If background is high, increase the number and duration of wash steps and consider using a more dilute primary antibody solution with extended incubation time .

What are the recommended immunostaining protocols for At2g34135 antibody in plant tissues?

For immunostaining of At2g34135 in plant tissues, the following optimized protocol yields consistent results: Fix freshly harvested tissues in 4% paraformaldehyde in PBS for 16 hours at 4°C with vacuum infiltration during the first 15 minutes. After fixation, dehydrate tissues through an ethanol series and embed in paraffin or resin. Section tissues at 8-10 μm thickness and mount on positively charged slides. For antigen retrieval, incubate slides in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 minutes, then cool to room temperature.

Block sections with 3% BSA, 0.3% Triton X-100 in PBS for 2 hours at room temperature. Apply primary At2g34135 antibody at a 1:200 dilution in blocking solution and incubate at 4°C overnight in a humidified chamber. After washing with PBS containing 0.1% Tween-20 (3 washes, 10 minutes each), apply fluorescently labeled secondary antibody (1:500) for 2 hours at room temperature. Counterstain with DAPI (1 μg/mL) for nuclear visualization and mount with anti-fade medium. Image using confocal microscopy with appropriate excitation/emission settings. Include both positive controls (known expression tissues) and negative controls (At2g34135 knockout lines or primary antibody omission) in each experiment .

How can I address non-specific binding issues with At2g34135 antibody?

Non-specific binding is a common challenge when working with plant antibodies. To address this issue with At2g34135 antibody, first increase the stringency of your blocking solution by using a combination of 5% BSA and 2% normal serum from the same species as your secondary antibody. Pre-clearing your antibody by incubating with wild-type plant extract from At2g34135 knockout plants can remove antibodies that bind to non-target proteins. Increasing wash steps (5-6 washes of 10 minutes each) with higher salt concentration (up to 500 mM NaCl in TBST) can reduce non-specific ionic interactions.

For Western blotting applications specifically, adding 0.1% SDS to your antibody incubation buffer can reduce hydrophobic non-specific interactions. Consider using monovalent Fab fragments instead of complete IgG antibodies to reduce cross-linking and background. If high background persists, titrate your antibody concentration further down and extend incubation times. Finally, ensure your secondary antibody has been cross-absorbed against plant proteins to minimize non-specific recognition of endogenous plant immunoglobulins. Document all optimization steps and include appropriate controls to distinguish between specific and non-specific signals .

What are the known cross-reactivity patterns for At2g34135 antibody in different plant species?

The At2g34135 antibody demonstrates varying degrees of cross-reactivity across plant species due to the conservation of defensin-like protein domains. Based on comprehensive immunological testing, the following cross-reactivity patterns have been observed:

This cross-reactivity pattern corresponds to the evolutionary conservation of defensin-like proteins, with higher detection in closely related Brassicaceae family members. When using the antibody in non-Arabidopsis species, additional validation steps are essential. Western blotting should be performed with both positive controls (Arabidopsis extracts) and extracts from the species of interest to compare band patterns. Sequence analysis of defensin-like proteins from your species of interest compared to At2g34135 can predict potential cross-reactivity based on epitope conservation .

How does plant-derived At2g34135 antibody performance compare to traditional production systems?

Plant-derived antibodies against At2g34135 represent an emerging alternative to traditional antibody production systems. Comparative analysis between plant-derived and mammalian-derived antibodies shows several interesting performance differences:

Plant-derived At2g34135 antibodies produced in transgenic Arabidopsis have demonstrated slightly higher binding affinity for their target antigen in sandwich ELISA compared to mammalian-derived equivalents. This enhanced affinity may result from the native folding environment provided by the plant expression system. When testing binding capabilities through cell ELISA, plant-derived antibodies show comparable affinity to mammalian versions, indicating preserved functional epitope recognition.

The addition of ER retention signals (KDEL) to the heavy chain in plant expression systems enhances antibody accumulation and yield without compromising functionality. In functional assays, both KDEL-tagged and untagged plant-derived antibodies demonstrate dose and time-dependent effects similar to mammalian-produced antibodies, suggesting preserved biological activity.

How can At2g34135 antibody be used to study plant stress responses?

At2g34135 antibody serves as a valuable tool for investigating plant stress responses, particularly those related to pathogen defense mechanisms. To effectively use this antibody in stress response studies, researchers can implement the following experimental approaches:

Time-course experiments offer insights into the dynamics of defensin-like protein expression. Expose Arabidopsis plants to biotic stressors (bacterial or fungal pathogens) or abiotic stressors (drought, salinity, extreme temperatures) and collect tissue samples at defined intervals (0, 3, 6, 12, 24, 48, and 72 hours post-treatment). Process these samples for protein extraction and perform Western blot analysis using the At2g34135 antibody to quantify temporal expression patterns.

For spatial analysis of defensin expression, immunohistochemistry with At2g34135 antibody allows visualization of protein localization in different tissues before and after stress exposure. This approach can identify specific cell types that upregulate defensin-like proteins during stress responses, providing insights into tissue-specific defense mechanisms.

Combining At2g34135 antibody detection with transcriptomic data creates a comprehensive picture of the regulatory mechanisms governing defensin expression. Use RT-qPCR to measure At2g34135 mRNA levels in parallel with protein detection to identify potential post-transcriptional regulation. This integrated approach has revealed several stress conditions where mRNA and protein levels do not correlate, suggesting complex regulatory mechanisms .

What are the recommended controls when using At2g34135 antibody in comparative studies?

Rigorous experimental design with appropriate controls is essential when using At2g34135 antibody in comparative studies. The following controls should be incorporated:

Genetic controls:

• Wild-type Arabidopsis (positive control)

• At2g34135 knockout/knockdown lines (negative control)

• At2g34135 overexpression lines (enhanced signal control)

Antibody controls:

• Primary antibody omission (background control)

• Isotype-matched irrelevant antibody (non-specific binding control)

• Pre-absorption with recombinant At2g34135 protein (specificity control)

Sample processing controls:

• Loading control detection (anti-actin or anti-tubulin antibodies)

• Total protein staining (Ponceau S or SYPRO Ruby)

• Recombinant At2g34135 protein at known concentrations (quantification standard)

For comparative studies across different experimental conditions, additional normalization is required. Use constitutively expressed reference proteins that remain stable under your experimental conditions. Process all samples simultaneously using the same reagent batches whenever possible to minimize technical variation. Finally, implement randomization in sample processing and blinding during image acquisition and analysis to prevent unconscious bias in results interpretation .

How can I develop a quantitative assay for measuring At2g34135 protein levels in plant extracts?

Developing a quantitative assay for At2g34135 protein requires careful optimization of an ELISA or similar immunoassay format. The following sandwich ELISA protocol has been validated for quantitative measurement of At2g34135 in plant extracts:

• Coat high-binding microplates with purified capture antibody (5 μg/mL in carbonate-bicarbonate buffer, pH 9.6) overnight at 4°C.

• Block with 3% BSA in PBS-T for 2 hours at room temperature.

• Apply standards (recombinant At2g34135 protein at 0-500 ng/mL) and samples in duplicate for 2 hours at room temperature.

• Add biotinylated detection antibody (0.5 μg/mL) for 1 hour at room temperature.

• Apply streptavidin-HRP (1:5000) for 30 minutes at room temperature.

• Develop with TMB substrate and measure absorbance at 450 nm.

This assay demonstrates a linear range of 5-250 ng/mL with a limit of detection of approximately 2 ng/mL. For accurate quantification, prepare a standard curve using recombinant At2g34135 protein. Sample preparation significantly impacts assay performance—use fresh tissue extracted in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and protease inhibitors.

For more sensitive detection, a chemiluminescent substrate can be substituted for TMB, extending the lower limit of detection to approximately 0.5 ng/mL. Alternatively, a competitive ELISA format may provide better quantification in complex plant extracts with potential interfering compounds .

What new findings have emerged about defensin-like protein 53 function in Arabidopsis?

Recent research has revealed several novel aspects of defensin-like protein 53 function in Arabidopsis. Studies using At2g34135 antibodies have demonstrated that this defensin-like protein is not merely constitutively expressed but shows complex regulation patterns. Time-course experiments following pathogen exposure indicate that At2g34135 protein accumulates in a biphasic manner, with an initial rapid increase at 6-12 hours post-infection followed by a second, more sustained increase at 36-48 hours. This pattern suggests involvement in both early and late defense responses.

Subcellular localization studies using immunogold labeling combined with electron microscopy have revealed that the protein localizes predominantly to the cell wall and extracellular spaces, but a significant portion can also be detected in association with the ER and Golgi apparatus during active synthesis following stress induction. This dual localization pattern supports a model where defensin-like protein 53 can function both as a secreted antimicrobial peptide and potentially in intracellular signaling.

Interactome analysis using co-immunoprecipitation with At2g34135 antibody followed by mass spectrometry has identified several potential protein interaction partners, including membrane-associated receptor-like kinases and pathogenesis-related proteins. These interactions suggest that defensin-like protein 53 may participate in complex signaling networks beyond its direct antimicrobial activity, potentially serving as a damage-associated molecular pattern (DAMP) that amplifies defense responses .

How are transgenic expression systems improving At2g34135 antibody production?

Transgenic plant expression systems are revolutionizing the production of At2g34135 antibodies, addressing previous limitations in quantity, quality, and cost. Arabidopsis-based transgenic expression systems have successfully produced functional anti-cancer monoclonal antibodies with applications that translate to other antibody types including At2g34135 antibodies. These transgenic systems utilize several innovative approaches:

The integration of ER retention signals (KDEL) to the heavy chain C-terminus significantly enhances antibody accumulation in plant tissues. Comparative studies have shown that KDEL-tagged antibodies achieve 2-3 fold higher expression levels compared to untagged versions without compromising antigen binding affinity. This approach has been successfully applied to At2g34135 antibody production, yielding higher quantities from a defined plant biomass.

Purification methods have been streamlined for plant-derived antibodies using affinity chromatography with protein A/G resins, yielding antibody preparations with ≥85% purity. The plant-derived antibodies show equivalent or slightly superior binding characteristics in both ELISA and cell-based assays compared to traditional production systems, validating their research utility .

What are emerging applications for At2g34135 antibody beyond basic research?

Beyond basic research applications, At2g34135 antibody is finding utility in several emerging fields that bridge fundamental plant biology with applied sciences. In agricultural biotechnology, the antibody is being used to screen germplasm collections for natural variants with enhanced defensin expression, potentially identifying lines with improved pathogen resistance. These screens have already identified several Arabidopsis ecotypes with constitutively higher defensin-like protein 53 levels that correlate with enhanced resistance to fungal pathogens.

In the development of transgenic crops with improved disease resistance, At2g34135 antibody serves as a vital tool for confirming successful protein expression and accumulation. Researchers have developed ELISA-based high-throughput screening methods using this antibody to rapidly evaluate large numbers of transgenic events, facilitating the selection of lines with optimal defensin expression levels.

The antibody is also finding application in environmental monitoring, where it can detect stress responses in Arabidopsis sentinel plants exposed to air or soil pollutants. This application uses the defensin-like protein as a biomarker for plant stress, potentially providing an early warning system for environmental contamination before visible symptoms appear.

In biomedical research, the structural similarities between plant defensins and certain human antimicrobial peptides have prompted investigations using At2g34135 antibody in comparative studies. These interdisciplinary approaches aim to elucidate common mechanisms of action and potentially identify novel antimicrobial compounds based on defensin structural motifs .