At5g61540 Antibody

What is the At5g61540 gene in Arabidopsis thaliana and what protein does it encode?

At5g61540 is a gene locus in the Arabidopsis thaliana genome, identified as part of chromosome 5. The gene encodes a protein with UniProt accession number Q56W64 . The "At" prefix indicates Arabidopsis thaliana, "5" refers to chromosome 5, and "g61540" represents the specific gene locus position. Understanding the gene's position and encoded protein is essential for planning experimental approaches that utilize antibodies against this target.

How should At5g61540 Antibody be stored and handled to maintain its efficacy?

For optimal maintenance of antibody function, store At5g61540 Antibody at -20°C for long-term storage and at 4°C for short-term use (up to one month). Avoid repeated freeze-thaw cycles, as these can significantly reduce antibody activity through protein denaturation. When handling, maintain sterile conditions and use appropriate pipetting techniques to prevent contamination. For working solutions, dilute in buffer containing a carrier protein (typically 1% BSA) and preserve with sodium azide (0.02%) if the solution will be stored for extended periods.

What controls should be included when using At5g61540 Antibody in immunoblotting experiments?

A methodologically sound experimental design for At5g61540 Antibody applications should include:

• Positive control: Wild-type Arabidopsis thaliana tissue expressing the target protein

• Negative control: Either knockout/knockdown plant lines for At5g61540 or tissue where the target protein is not expressed

• Loading control: Detection of a housekeeping protein (such as actin or tubulin) to normalize protein loading

• Pre-immune serum control: To identify non-specific binding

• Competing peptide control: Pre-incubation of antibody with the immunizing peptide should abolish specific binding

These controls help validate antibody specificity and ensure accurate interpretation of experimental results.

What are the recommended dilutions for At5g61540 Antibody in different applications?

Optimal dilutions should be determined empirically for each specific application and experimental condition.

How can I validate the specificity of At5g61540 Antibody for my particular research application?

Validating antibody specificity requires a multi-faceted approach:

• Western blot analysis comparing wild-type to knockout/knockdown lines: A specific antibody will show reduced or absent signal in genetic knockout lines.

• Mass spectrometry validation: Immunoprecipitate the target protein using the At5g61540 Antibody and confirm identity through mass spectrometry.

• Epitope mapping: Determine the exact binding region of the antibody and assess potential cross-reactivity with related proteins.

• Heterologous expression: Test antibody against recombinant Q56W64 protein expressed in a bacterial or insect cell system.

• Immunodepletion: Pre-adsorb the antibody with purified target protein to demonstrate that this eliminates specific binding.

This systematic validation approach ensures that experimental results are attributable to specific binding rather than cross-reactivity or non-specific interactions.

What are the best approaches for optimizing immunoprecipitation protocols using At5g61540 Antibody?

Immunoprecipitation with plant proteins requires careful optimization due to the complex plant matrix and presence of interfering compounds. For At5g61540 Antibody:

• Extraction buffer optimization: Test different extraction buffers containing various detergents (CHAPS, Triton X-100, NP-40) at different concentrations (0.1-1%).

• Crosslinking considerations: For transient or weak interactions, implement chemical crosslinking with formaldehyde (1%) or DSP (dithiobis(succinimidyl propionate)).

• Pre-clearing strategy: Implement extensive pre-clearing with Protein A/G beads to reduce non-specific binding.

• Antibody coupling: For reproducible results, covalently couple At5g61540 Antibody to beads using dimethyl pimelimidate or similar crosslinkers.

• Elution conditions: Compare different elution methods (low pH, competing peptide, SDS) for yield and maintenance of protein activity.

• Quantitative assessment: Use quantitative mass spectrometry with isotope labeling to assess enrichment efficiency.

This methodical optimization ensures maximum recovery of the target protein while minimizing contaminants.

How can At5g61540 Antibody be adapted for chromatin immunoprecipitation (ChIP) studies in Arabidopsis?

Adapting the antibody for ChIP requires specialized considerations:

• Crosslinking protocol: Optimize formaldehyde concentration (typically 1-3%) and fixation time (10-20 minutes) specifically for Arabidopsis tissue.

• Sonication parameters: Determine optimal sonication conditions to generate 200-500bp DNA fragments from plant chromatin.

• Antibody binding: Perform titration experiments (5-10μg antibody per ChIP reaction) to determine the optimal antibody:chromatin ratio.

• Pre-blocking strategy: Pre-block the antibody with competing non-specific proteins to reduce background.

• Sequential ChIP: For analyzing co-occupancy with other factors, implement sequential ChIP protocols with appropriate controls.

• ChIP-seq validation: Confirm enrichment of specific genomic regions via qPCR before proceeding to genome-wide sequencing.

Successful ChIP experiments with At5g61540 Antibody would provide insights into potential DNA-binding properties or chromatin association of the target protein.

What approaches can resolve contradictory results when using At5g61540 Antibody across different experimental platforms?

When facing contradictory results:

• Epitope accessibility analysis: Different experimental conditions may affect epitope exposure. Implement alternative extraction methods, denaturation conditions, or fixation protocols.

• Post-translational modification interference: Test whether phosphorylation, ubiquitination, or other modifications affect antibody recognition using phosphatase treatment or deubiquitinating enzymes.

• Isoform specificity evaluation: Determine if the antibody recognizes all protein isoforms by comparing results with RNA-seq data and targeted expression analysis.

• Batch variation assessment: Compare antibody lots using quantitative assays and establish internal reference standards.

• Method-specific interference: Identify potential interfering agents specific to each method and implement appropriate blocking or pre-adsorption steps.

• Computational validation: Use bioinformatics to cross-reference results with available proteomics databases, expression atlases, and protein interaction networks.

This systematic troubleshooting allows identification of method-specific artifacts versus true biological phenomena.

How should tissue samples be prepared for optimal detection of At5g61540 protein in Arabidopsis?

Effective tissue preparation is critical for antibody-based detection:

• Harvesting timing: Collect tissue at the developmental stage where the target protein is most abundant based on transcriptomic data.

• Tissue stabilization: Flash-freeze samples in liquid nitrogen immediately after collection to prevent protein degradation.

• Extraction buffer composition: For plant tissues, use a buffer containing:

  • 50mM Tris-HCl (pH 7.5)

  • 150mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail specific for plant tissues

  • 1mM PMSF

  • 10mM DTT to reduce oxidation

• Mechanical disruption: Grind tissue thoroughly in liquid nitrogen using a mortar and pestle before adding extraction buffer.

• Clarification: Centrifuge at 12,000g for 15 minutes at 4°C and collect supernatant.

• Protein concentration: Standardize protein concentration using Bradford or BCA assay before immunological applications.

This methodical preparation ensures maximum protein yield while preserving epitope integrity for antibody recognition.

What are the critical parameters for successful immunolocalization of At5g61540 protein in plant tissues?

Immunolocalization in plant tissues presents unique challenges:

• Fixation optimization: Compare paraformaldehyde (4%) versus glutaraldehyde/paraformaldehyde combinations to balance structural preservation with epitope accessibility.

• Cell wall considerations: Implement cell wall digestion using enzymes (pectolyase, cellulase) or include appropriate detergents to enhance antibody penetration.

• Permeabilization protocol: Test graduated methanol series versus detergent-based permeabilization to determine optimal conditions.

• Blocking strategy: Use plant-specific blocking solutions containing:

  • 5% normal serum from the secondary antibody host species

  • 3% BSA

  • 0.1% plant-derived glycoproteins

  • 0.05% Tween-20

• Antibody incubation: Extend primary antibody incubation to 48-72 hours at 4°C for thick plant sections.

• Signal amplification: Consider tyramide signal amplification if target protein is low-abundance.

• Autofluorescence management: Implement specific treatments (sodium borohydride, Sudan Black B) to reduce plant autofluorescence.

This comprehensive approach maximizes signal-to-noise ratio in plant immunolocalization experiments.

How can At5g61540 Antibody be utilized in protein-protein interaction studies?

For investigating protein interaction networks:

• Co-immunoprecipitation strategy: Optimize buffer conditions (salt concentration, detergent type/concentration) to preserve native interactions during extraction.

• Proximity ligation assay (PLA) adaptation: Combine At5g61540 Antibody with antibodies against suspected interaction partners in fixed Arabidopsis tissues.

• Bimolecular Fluorescence Complementation (BiFC) correlation: Validate antibody-based interaction findings with orthogonal BiFC experiments.

• FRET-FLIM analysis: Use the antibody for immunolocalization in conjunction with FRET-FLIM measurements to correlate subcellular localization with interaction zones.

• Pull-down validation: Compare immunoprecipitation results with pull-down assays using recombinant tagged versions of the protein.

• Crosslinking mass spectrometry integration: Combine chemical crosslinking with immunoprecipitation and mass spectrometry for identifying interaction interfaces.

This multi-technique approach provides robust validation of protein-protein interactions.

What are common causes of high background when using At5g61540 Antibody and how can they be mitigated?

High background issues can be systematically addressed:

• Non-specific binding: Increase blocking agent concentration (5-10% BSA or normal serum) and extend blocking time (2-3 hours at room temperature).

• Secondary antibody cross-reactivity: Pre-adsorb secondary antibody with plant tissue extract from the same species.

• Endogenous peroxidase/phosphatase activity: Include specific inhibitors in the protocol:

  • For HRP-based detection: 0.3% H₂O₂ in methanol for 30 minutes

  • For AP-based detection: 1mM levamisole

• Insufficient washing: Extend wash steps (5 x 10 minutes) and increase detergent concentration in wash buffers (0.1-0.3% Tween-20).

• Antibody concentration: Perform titration experiments to determine optimal concentration that maximizes specific signal while minimizing background.

• Plant-specific compounds: Add polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP) to extraction and blocking buffers to sequester phenolic compounds.

How can the quality and activity of At5g61540 Antibody be assessed over time and storage?

To monitor antibody quality:

• Activity tracking: Establish a reference Western blot protocol using a standard positive control sample and perform at regular intervals.

• Titer determination: Conduct ELISA-based titration experiments to quantify binding activity over time.

• Epitope recognition test: Use a synthetic peptide array to measure epitope-specific binding capacity.

• Functional assay: If the antibody has neutralizing activity, monitor this property using appropriate functional assays.

• Physical assessment: Check for visible precipitation, turbidity, or color changes that may indicate antibody degradation.

• Storage validation: Compare aliquots stored under different conditions to determine optimal preservation methods.

Implement a quality control schedule with defined acceptance criteria to ensure experimental reproducibility over extended research periods.

How can At5g61540 Antibody be adapted for high-throughput screening applications?

For scaling to high-throughput formats:

• Microplate optimization: Determine minimum antibody concentration effective in 384-well format ELISA.

• Automation compatibility: Assess antibody performance with automated liquid handling systems, focusing on:

  • Stability in robotic sample preparation conditions

  • Reproducibility across plate positions

  • Edge effects mitigation strategies

• Multiplex adaptation: Test compatibility with multiplexed detection platforms like Luminex or protein arrays.

• Miniaturization strategy: Validate antibody function in reduced reaction volumes (5-10μL) and modified surface chemistries.

• Signal detection: Optimize signal amplification methods for automated plate readers:

  • Chemiluminescence parameters

  • Fluorescence wavelength optimization

  • Colorimetric endpoint stability

• Data normalization: Establish internal standards and normalization algorithms specific to plant extract complexity.

This systematic approach enables reliable high-content screening while maintaining detection sensitivity.

What considerations are important when using At5g61540 Antibody for quantitative proteomics?

For quantitative applications:

• Standard curve development: Generate calibration curves using purified recombinant Q56W64 protein.

• Dynamic range determination: Establish the linear detection range for the antibody in complex plant extracts.

• Matrix effect evaluation: Assess how different plant tissues and extraction methods affect quantification accuracy.

• Normalization strategy: Implement appropriate normalization to housekeeping proteins that remain stable under experimental conditions.

• Absolute quantification: Consider developing isotope-labeled internal standards (AQUA peptides) corresponding to the antibody's epitope region.

• Statistical validation: Establish minimum technical and biological replicate numbers required for reliable quantification.

• Cross-platform validation: Verify antibody-based quantification against orthogonal methods like MRM (Multiple Reaction Monitoring) mass spectrometry.

These considerations ensure that quantitative measurements accurately reflect biological reality rather than technical artifacts.

How should At5g61540 Antibody data be integrated with other omics datasets in Arabidopsis research?

Effective multi-omics integration requires:

• Transcriptomic correlation: Compare protein detection levels with RNA-seq or microarray expression data for the At5g61540 gene across developmental stages and conditions.

• Proteomics validation: Cross-reference antibody-based detection with mass spectrometry proteomics data to confirm protein abundance patterns.

• Metabolomic association: Correlate protein levels with relevant metabolite profiles to establish functional relationships.

• Phenotypic mapping: Link protein expression patterns detected by the antibody to phenotypic data from mutant lines.

• Pathway contextualization: Place protein detection data within relevant biochemical and signaling pathways using tools like AraCyc or STRING.

• Data visualization: Implement integrative visualization approaches that place antibody-derived data in broader biological context.

This integrative approach maximizes the biological insights gained from antibody-based experiments by providing mechanistic context and functional validation.

What are best practices for reproducibility when publishing research using At5g61540 Antibody?

To ensure research reproducibility:

• Antibody documentation: Report complete antibody information:

  • Catalog number (CSB-PA681975XA01DOA)

  • Lot number

  • Host species

  • Clonality

  • Epitope sequence

  • Supplier (Cusabio)

• Validation evidence: Include antibody validation data demonstrating specificity in supplementary materials.

• Protocol transparency: Provide detailed protocols including:

  • Blocking conditions (agent, concentration, time)

  • Antibody dilutions and incubation parameters

  • Washing procedures

  • Detection methods

• Control implementation: Clearly describe all positive and negative controls used.

• Image acquisition: Document complete imaging parameters for microscopy or blot scanning.

• Quantification methodology: Detail image analysis software, settings, and statistical approaches.

• Data availability: Deposit raw image data in appropriate repositories following FAIR principles.

Adherence to these practices ensures that research using At5g61540 Antibody can be evaluated and reproduced by the scientific community.