At5g51845 Antibody

What is the At5g51845 gene and its function in Arabidopsis thaliana?

At5g51845 encodes a defensin-like (DEFL) family protein in Arabidopsis thaliana. Defensin-like proteins comprise a large family of small cysteine-rich proteins that play critical roles in plant immunity and defense mechanisms against pathogens . The DEFL family in Arabidopsis includes numerous members with diverse functions, primarily involved in antimicrobial activity and signaling processes during plant development and stress responses.

To study this protein effectively, researchers should consider its structural characteristics, including conserved cysteine residues that form disulfide bridges essential for protein stability and function. Experimental approaches for functional characterization may include gene expression analysis under various stress conditions, protein localization studies, and phenotypic analysis of knockout or overexpression lines.

What technical specifications should researchers know about the At5g51845 antibody?

The At5g51845 antibody is a rabbit polyclonal antibody produced against recombinant Arabidopsis thaliana At5g51845 protein . This affinity-purified antibody is designed primarily for plant-based research with validated applications in ELISA and Western blot techniques . The product typically includes:

The antibody recognizes plant species and should be stored at -20°C or -80°C for optimal stability and performance . When designing experiments, researchers should consider the polyclonal nature of this antibody, which provides broad epitope recognition but may require additional optimization steps for specific applications.

How should researchers validate the specificity of the At5g51845 antibody?

Validating antibody specificity is crucial for obtaining reliable research results. For At5g51845 antibody, implement a comprehensive validation strategy that includes:

• Positive and negative controls: Use the provided recombinant antigen (200μg) as a positive control and pre-immune serum (1ml) as a negative control in initial validation experiments .

• Western blot analysis: Run parallel samples from wild-type plants and At5g51845 knockout/knockdown mutants. A specific antibody will show reduced or absent signal in mutant lines.

• Peptide competition assay: Pre-incubate the antibody with excess recombinant At5g51845 protein before immunodetection. Specific binding should be significantly reduced or eliminated.

• Cross-reactivity assessment: Test the antibody against closely related DEFL family proteins to evaluate potential cross-reactivity, especially important given the sequence similarities within this protein family.

• Immunoprecipitation followed by mass spectrometry: This approach can definitively identify the proteins being recognized by the antibody in complex biological samples.

Document all validation steps methodically, as antibody specificity is fundamental to result interpretation and reproducibility in plant molecular biology research.

What are the optimal sample preparation protocols for At5g51845 detection?

Effective sample preparation is critical for detecting At5g51845 protein. For plant tissue extraction, consider these methodological recommendations:

• Tissue selection: Choose appropriate tissues based on At5g51845 expression patterns. For defensin-like proteins, young leaves, roots, or tissues under pathogen stress often show higher expression levels.

• Extraction buffer optimization:

  • For soluble proteins: Use 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM EDTA, 10% glycerol, 1mM DTT, and protease inhibitor cocktail

  • For membrane-associated proteins: Add 0.5-1% non-ionic detergent (e.g., NP-40 or Triton X-100)

• Protein denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing a reducing agent for Western blot applications.

• Protein concentration determination: Use Bradford or BCA assay to standardize loading amounts.

• Sample storage: Prepare aliquots to avoid freeze-thaw cycles and store at -80°C.

For antibody-based detection techniques, optimize protein loading (typically 20-50μg total protein per lane for Western blot) and antibody concentration (start with a 1:1000 dilution and adjust as needed based on signal-to-noise ratio).

What controls should be included when working with the At5g51845 antibody?

Implementing appropriate controls is essential for experimental rigor when working with At5g51845 antibody:

For quantitative analyses, include a dilution series of recombinant protein to create a standard curve. Additionally, consider tissue-specific controls, particularly when examining tissues with varying protein expression levels or potential post-translational modifications.

What are the best approaches for optimizing Western blot protocols with At5g51845 antibody?

Western blot optimization for At5g51845 detection requires systematic adjustment of multiple parameters:

• Protein extraction optimization:

  • Test different extraction buffers with varying detergent concentrations

  • Compare mechanical disruption methods (e.g., grinding in liquid nitrogen vs. bead beating)

  • Evaluate protein precipitation methods if target concentration is low

• Gel electrophoresis parameters:

  • Select appropriate acrylamide percentage (12-15% recommended for small defensin-like proteins)

  • Consider gradient gels for better resolution

  • Optimize running conditions (voltage, time) to prevent protein degradation

• Transfer optimization:

  • For small proteins like defensin-like family members, use PVDF membranes with 0.2μm pore size

  • Consider semi-dry transfer for 20-30 minutes or wet transfer with 10-20% methanol

  • Use chilled transfer buffer containing SDS (0.01-0.02%) to facilitate small protein transfer

• Blocking and antibody incubation:

  • Test different blocking agents (5% non-fat milk vs. 3-5% BSA)

  • Optimize primary antibody dilution (typically start at 1:1000 and adjust)

  • Determine optimal incubation time and temperature (4°C overnight vs. room temperature for 1-2 hours)

• Signal detection optimization:

  • Compare different detection systems (chemiluminescence vs. fluorescence)

  • For low abundance targets, consider signal amplification methods

Implement a systematic grid testing approach, modifying one variable at a time to identify optimal conditions for detecting At5g51845.

How can researchers use At5g51845 antibody for studying protein-protein interactions?

The At5g51845 antibody can be instrumental in studying protein-protein interactions involving defensin-like proteins through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Cross-link proteins in vivo using formaldehyde (0.5-1%) for 10-15 minutes

  • Prepare lysates under non-denaturing conditions to preserve protein complexes

  • Immobilize At5g51845 antibody on protein A/G beads (5-10μg antibody per 50μl bead slurry)

  • Incubate with lysate, wash stringently, and elute for downstream analysis

  • Identify interaction partners through mass spectrometry or immunoblotting

• Proximity Ligation Assay (PLA):

  • Fix plant tissues with 4% paraformaldehyde

  • Incubate with At5g51845 antibody and antibody against suspected interaction partner

  • Follow with PLA-specific secondary antibodies and ligation/amplification steps

  • Quantify interaction signals using confocal microscopy

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Clone At5g51845 and candidate interactor genes into BiFC vectors

  • Transform protoplasts or generate stable transgenic plants

  • Visualize protein interactions through reconstituted fluorescent protein signals

  • Use antibody for parallel confirmation of protein expression levels

• Pull-down assays with recombinant proteins:

  • Express epitope-tagged recombinant At5g51845 protein

  • Use At5g51845 antibody to confirm expression and purification efficiency

  • Perform pull-down experiments with plant lysates

  • Identify specific interactions through comparative analysis with control pull-downs

When reporting protein interaction data, always include appropriate controls and multiple complementary methods for validation.

What methodological considerations are important for immunolocalization of At5g51845 protein?

Immunolocalization of At5g51845 requires careful attention to tissue preparation, fixation, and detection protocols:

• Tissue preparation:

  • Fix fresh tissues in 4% paraformaldehyde in PBS or PEM buffer for 2-4 hours

  • For better antibody penetration, consider 1-2% glutaraldehyde addition for certain applications

  • Optimize fixation time: excessive fixation may mask epitopes, insufficient fixation may compromise tissue integrity

  • Process fixed tissues through ethanol series and embedding medium appropriate for intended sectioning method

• Antigen retrieval methods:

  • Test heat-induced epitope retrieval (pressure cooker method with citrate buffer, pH 6.0)

  • Compare with enzymatic retrieval using proteases (proteinase K at 10-20μg/ml for 10-15 minutes)

  • Document optimal retrieval conditions for specific tissues

• Immunolabeling protocol:

  • Block with 2-5% BSA, 5-10% normal serum in PBS with 0.1-0.3% Triton X-100

  • Incubate with At5g51845 antibody at optimized dilution (1:100-1:500 range for immunohistochemistry)

  • For fluorescence detection, use appropriate fluorophore-conjugated secondary antibodies

  • Include parallel negative controls (pre-immune serum, secondary antibody only)

• Special considerations for plant tissues:

  • Address autofluorescence using Sudan Black B (0.1-0.3% in 70% ethanol) for 10-30 minutes

  • Consider alternative clearance methods for thick tissues

  • For higher resolution, evaluate super-resolution microscopy techniques

For quantitative analysis of localization patterns, develop standardized imaging parameters and analysis workflows using appropriate software tools.

How can At5g51845 antibody be used to study the protein's role in plant stress responses?

Investigating At5g51845's role in plant stress responses requires integrated experimental approaches:

• Stress-induced expression analysis:

  • Subject plants to relevant stresses (pathogen infection, drought, salinity)

  • Collect tissues at different time points post-stress induction

  • Use At5g51845 antibody in Western blot to quantify protein levels

  • Normalize against appropriate loading controls

  • Correlate protein levels with transcript abundance through parallel RT-qPCR

• Spatial regulation under stress:

  • Perform immunolocalization of At5g51845 in stressed vs. control tissues

  • Document changes in subcellular localization or tissue distribution

  • Quantify signal intensity changes across different cell types

• Protein modification analysis:

  • Investigate post-translational modifications using specialized techniques:

    • Phosphorylation: Phos-tag gels followed by Western blot with At5g51845 antibody

    • Glycosylation: Treat samples with glycosidases before immunoblotting

    • Ubiquitination: Immunoprecipitate with At5g51845 antibody and probe with anti-ubiquitin

• Functional protein complex dynamics:

  • Compare protein interaction partners under normal vs. stress conditions

  • Use co-immunoprecipitation with At5g51845 antibody followed by mass spectrometry

  • Validate key interactions through reciprocal co-IP or BiFC

• Transgenic approaches with integrated antibody validation:

  • Generate overexpression and knockdown/knockout lines of At5g51845

  • Use the antibody to confirm altered protein levels

  • Assess phenotypic consequences under various stress conditions

  • Correlate molecular and physiological parameters

Document stress treatment conditions precisely, including intensity, duration, and environmental parameters to ensure experimental reproducibility.

What are effective strategies for multiplexing At5g51845 antibody with other antibodies?

Multiplexing the At5g51845 antibody with other antibodies enables simultaneous detection of multiple proteins, providing valuable insights into co-localization and relative expression patterns:

• Western blot multiplexing approaches:

  • Sequential immunoblotting: Strip and reprobe membranes with careful validation of stripping efficiency

  • Dual-color detection: Use At5g51845 antibody with antibodies raised in different host species

  • Size-based multiplexing: Detect proteins of sufficiently different molecular weights on the same blot

• Immunofluorescence multiplexing:

  • Primary antibody selection: Combine At5g51845 rabbit polyclonal with mouse or goat antibodies against other targets

  • Secondary antibody selection: Use spectrally distinct fluorophores with minimal overlap

  • Sequential detection protocol:

  Step	Procedure	Duration
  1	Incubate with first primary antibody (At5g51845)	Overnight, 4°C
  2	Wash extensively (3-5x with PBST)	30-45 minutes
  3	Apply first secondary antibody	1-2 hours, RT
  4	Wash extensively	30-45 minutes
  5	Apply second primary antibody	Overnight, 4°C
  6	Wash extensively	30-45 minutes
  7	Apply second secondary antibody	1-2 hours, RT
  8	Final washing and mounting	45-60 minutes

• Validation of multiplexed detection:

  • Perform single-antibody controls in parallel

  • Include absorption controls to verify absence of cross-reactivity

  • Document potential spectral bleed-through using single-fluorophore samples

  • Analyze co-localization using appropriate quantitative metrics (Pearson's correlation, Manders' coefficients)

• Advanced multiplexing technologies:

  • Consider tyramide signal amplification for detecting low-abundance proteins

  • Evaluate sequential immunofluorescence techniques for detecting multiple rabbit antibodies

  • For mass cytometry applications, conjugate At5g51845 antibody with distinct metal isotopes

When reporting multiplexed detection results, thoroughly document all experimental controls and optimization steps to ensure result reliability.

How should researchers address weak or absent signals when using At5g51845 antibody?

When encountering weak or absent signals with At5g51845 antibody, implement a systematic troubleshooting approach:

• Expression level considerations:

  • Verify At5g51845 expression in your experimental system through RT-qPCR

  • Consider tissue-specific or developmental regulation of the target protein

  • Evaluate induction conditions that might increase protein levels

• Sample preparation optimization:

  • Test multiple protein extraction protocols with different detergents and buffer compositions

  • Evaluate protein enrichment methods (e.g., immunoprecipitation before Western blot)

  • Ensure complete solubilization of membrane-associated proteins if applicable

  • Add protease inhibitors freshly before extraction

• Detection sensitivity enhancement:

  • Increase protein loading (up to 50-80μg total protein per lane)

  • Decrease antibody dilution (1:500 or 1:250 instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try more sensitive detection systems (enhanced chemiluminescence, fluorescent detection)

  • Consider signal amplification methods (biotin-streptavidin systems)

• Epitope accessibility improvement:

  • Test different antigen retrieval methods for immunohistochemistry

  • Evaluate alternative membrane blocking agents (5% BSA vs. 5% milk)

  • Try different membrane types (PVDF vs. nitrocellulose)

• Antibody functionality verification:

  • Test antibody with the provided positive control (recombinant protein, 200μg)

  • Check antibody storage conditions and avoid repeated freeze-thaw cycles

  • Consider freshly ordering antibody if the current lot is old

Document all troubleshooting steps methodically to identify the specific variables affecting detection sensitivity.

What approaches can address non-specific binding or high background with At5g51845 antibody?

High background or non-specific binding can significantly impact data quality. Address these issues through:

• Blocking optimization:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blocking buffers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to all washing and antibody incubation buffers

• Antibody dilution optimization:

  • Perform a dilution series (1:500 to 1:5000) to identify optimal concentration

  • Pre-absorb antibody with proteins from non-target species if cross-reactivity is suspected

  • Purify antibody through antigen-specific affinity chromatography for critical applications

• Washing protocol enhancement:

  • Increase washing duration and frequency (5-6 washes of 10 minutes each)

  • Use higher stringency wash buffers (increase salt concentration to 300-500mM NaCl)

  • Add 0.1% SDS to wash buffer for particularly problematic backgrounds

• Cross-reactivity reduction:

  • Use the pre-immune serum provided with the antibody (1ml) to identify non-specific binding patterns

  • Consider peptide competition assays to distinguish specific from non-specific signals

  • For immunohistochemistry, include tissue-specific negative controls

• Detection system optimization:

  • For chemiluminescence, reduce substrate incubation time

  • For fluorescence detection, include additional washing steps after secondary antibody incubation

  • Optimize imaging parameters to balance specific signal detection and background reduction

Always include appropriate negative controls in parallel experiments to facilitate accurate interpretation of results.

How can researchers accurately quantify At5g51845 protein levels?

Accurate protein quantification requires rigorous methodological approaches:

• Western blot quantification:

  • Use a standard curve of recombinant At5g51845 protein (spanning the expected concentration range)

  • Include multiple biological and technical replicates (minimum n=3)

  • Select appropriate loading controls (constitutively expressed proteins unaffected by experimental conditions)

  • Apply densitometric analysis with background subtraction

  • Verify the linear range of detection for your system

• ELISA development:

  • Establish a sandwich ELISA using At5g51845 antibody as capture or detection antibody

  • Generate a standard curve using recombinant protein (provided with antibody, 200μg)

  • Optimize blocking, washing, and detection parameters

  • Validate reproducibility across multiple plates and days

• Quantitative considerations:

  • Normalize against total protein concentration determined by Bradford or BCA assay

  • For relative quantification, calculate fold changes relative to appropriate control samples

  • For absolute quantification, use purified recombinant protein standards

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Report both biological and technical variability

  • Consider power analysis to determine adequate sample size

• Advanced quantitative approaches:

  • For greater precision, consider targeted proteomics approaches (Selected Reaction Monitoring)

  • Implement fluorescence-based quantification using labeled secondary antibodies

  • For spatial quantification, develop immunofluorescence intensity measurement protocols

Document all quantification parameters, including image acquisition settings, analysis software, and calculation methods to ensure reproducibility.

How can At5g51845 antibody be adapted for high-throughput screening applications?

Adapting At5g51845 antibody for high-throughput applications requires optimization of protocols for automation and scalability:

• Microplate-based assay development:

  • Optimize ELISA protocols for 384-well format

  • Develop homogeneous assay formats to reduce washing steps

  • Establish robust positive and negative controls for plate normalization

  • Implement automated liquid handling systems for consistent reagent delivery

• Tissue microarray applications:

  • Develop miniaturized immunohistochemistry protocols

  • Optimize antibody concentration and incubation conditions for small tissue sections

  • Establish automated image acquisition and analysis pipelines

  • Validate signal consistency across the microarray

• Flow cytometry adaptation:

  • Develop cell permeabilization protocols compatible with intracellular At5g51845 detection

  • Optimize antibody concentration for flow cytometry applications

  • Establish appropriate gating strategies and controls

  • Consider fluorescent cell barcoding for multiplexed analysis

• Automated Western blot systems:

  • Adapt protocols for capillary-based protein separation systems

  • Optimize protein loading, antibody dilution, and incubation parameters

  • Develop standard curves for quantitative analysis

  • Validate across multiple sample types

• Quality control considerations:

  • Implement robust statistical methods for assay validation (Z'-factor calculation)

  • Establish acceptance criteria for high-throughput screening campaigns

  • Develop secondary confirmation assays for primary hits

Document optimization parameters methodically to ensure reproducibility across multiple experimental batches and operators.

What are the considerations for using At5g51845 antibody in single-cell protein analysis?

Single-cell protein analysis with At5g51845 antibody presents unique methodological challenges:

• Sample preparation optimization:

  • Develop gentle cell isolation protocols to preserve protein integrity

  • Optimize fixation and permeabilization conditions for single plant cells

  • Establish sorting parameters for specific cell populations if using flow cytometry

• Imaging-based single-cell analysis:

  • Implement high-resolution confocal microscopy with deconvolution

  • Optimize immunofluorescence protocols for maximum sensitivity

  • Develop automated image segmentation algorithms to identify individual cells

  • Establish quantitative parameters for single-cell protein expression analysis

• Mass cytometry applications:

  • Conjugate At5g51845 antibody with appropriate metal isotopes

  • Optimize staining protocols for plant cell suspensions

  • Develop appropriate gating and clustering strategies

  • Integrate with other protein markers for comprehensive phenotyping

• Validation approaches:

  • Correlate protein detection with single-cell RNA sequencing data

  • Implement spike-in controls for technical variation assessment

  • Develop computational methods to account for technical artifacts

• Data analysis considerations:

  • Apply dimensionality reduction techniques (t-SNE, UMAP) for visualization

  • Implement clustering algorithms to identify cell subpopulations

  • Develop trajectory inference methods for developmental studies

  • Establish appropriate statistical methods for single-cell protein quantification

When reporting single-cell analysis results, thoroughly document all preprocessing steps, quality control metrics, and analysis parameters to ensure reproducibility.

How can researchers integrate At5g51845 antibody-based detection with other -omics approaches?

Integrating antibody-based detection with multi-omics approaches provides comprehensive insights into biological systems:

• Proteogenomic integration:

  • Correlate At5g51845 protein levels (detected by antibody) with transcript abundance

  • Develop sampling protocols that enable parallel protein and RNA extraction

  • Apply computational methods to integrate protein and transcript data

  • Investigate potential post-transcriptional regulation mechanisms

• Spatial proteomics approaches:

  • Combine immunolocalization with in situ RNA hybridization

  • Develop multiplexed detection protocols for spatial co-expression analysis

  • Implement computational image analysis for quantitative spatial correlation

  • Consider emerging technologies like Spatial Transcriptomics with immunofluorescence

• Interaction proteomics:

  • Use At5g51845 antibody for immunoprecipitation followed by mass spectrometry

  • Develop crosslinking protocols to capture transient interactions

  • Implement quantitative approaches (SILAC, TMT) for comparative interaction studies

  • Validate key interactions through orthogonal methods

• Functional genomics integration:

  • Combine CRISPR-based gene editing with antibody-based protein detection

  • Develop inducible expression systems with quantitative protein analysis

  • Establish phenotypic readouts that correlate with protein expression levels

  • Implement systems biology approaches to model protein function

• Data integration frameworks:

  • Develop computational pipelines for multi-omics data integration

  • Implement statistical methods for correlation analysis across data types

  • Consider machine learning approaches for pattern recognition

  • Establish visualization tools for integrated data presentation

Document all experimental protocols and computational methods in sufficient detail to enable reproducibility of the integrated analysis workflow.

What are the key considerations for ensuring reproducible results with At5g51845 antibody?

Ensuring reproducibility with At5g51845 antibody requires meticulous attention to multiple experimental parameters:

By implementing these comprehensive reproducibility practices, researchers can enhance the reliability and impact of their At5g51845 antibody-based research in plant molecular biology.

What future developments might enhance At5g51845 antibody applications in plant research?

The future of At5g51845 antibody applications in plant research will likely be shaped by several emerging technologies and methodological advances:

• Antibody engineering advancements:

  • Development of recombinant antibody fragments with enhanced specificity

  • Creation of nanobodies or single-domain antibodies for improved tissue penetration

  • Implementation of site-specific conjugation strategies for optimal orientation

  • Generation of bispecific antibodies for simultaneous detection of multiple targets

• Advanced imaging technologies:

  • Integration with super-resolution microscopy for subcellular localization

  • Application in expansion microscopy for enhanced spatial resolution

  • Development of cleared-tissue immunolabeling for whole-organ imaging

  • Implementation of light-sheet microscopy for dynamic protein tracking

• Quantitative proteomics integration:

  • Development of improved targeted proteomics workflows with antibody-based enrichment

  • Implementation of multiplexed protein quantification using mass cytometry

  • Creation of spatial proteomics approaches combining antibody detection with mass spectrometry

  • Advancement of single-molecule detection methods for absolute quantification

• Translational applications:

  • Utilization in crop improvement programs targeting defensin-like proteins

  • Application in biosensor development for agricultural monitoring

  • Implementation in high-throughput phenotyping platforms

  • Integration into diagnostic tools for plant disease detection

• Computational and AI-driven advances:

  • Development of machine learning algorithms for automated image analysis

  • Creation of predictive models for protein-protein interactions

  • Implementation of systems biology approaches integrating antibody-derived data

  • Advancement of knowledge bases specific to plant immunity proteins