At2g39415 Antibody

What is the At2g39415 gene in Arabidopsis thaliana and what protein does it encode?

At2g39415 is a gene located on chromosome 2 of Arabidopsis thaliana, encoding a protein identified by UniProt accession number Q3EBJ8. This gene is part of the extensive genomic framework of Arabidopsis, which serves as a model organism for plant molecular and genetic research. Understanding this gene's function contributes to broader knowledge of plant developmental processes and stress responses. When designing experiments with the corresponding antibody, it's essential to consider the protein's predicted structure, expression patterns, and functional domains to appropriately interpret results.

What are the recommended applications for the At2g39415 Antibody?

The At2g39415 Antibody (CSB-PA666902XA01DOA) is suitable for several experimental applications in plant molecular biology research. These typically include:

Researchers should validate these applications with appropriate controls specific to their experimental design. The antibody is available in both concentrated (0.1ml) and standard (2ml) preparations to accommodate different experimental scales .

How should I prepare Arabidopsis samples for optimal antibody recognition?

Proper sample preparation is crucial for successful At2g39415 antibody applications. For protein extraction:

• Harvest fresh plant material (preferably young leaves or seedlings) and immediately flash-freeze in liquid nitrogen

• Grind tissue to a fine powder while keeping frozen

• Extract proteins using a buffer containing:

  • 50mM Tris-HCl (pH 7.5)

  • 150mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • 1mM EDTA

  • Protease inhibitor cocktail

• Centrifuge at 14,000g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration

• For Western blotting, denature samples at 95°C for 5 minutes in SDS sample buffer

This methodology ensures protein integrity while minimizing degradation and maximizing antibody recognition potential.

What controls should I include when working with the At2g39415 Antibody?

Rigorous experimental design requires appropriate controls when working with antibodies. For At2g39415 Antibody research, include:

• Positive control: Protein extract from wild-type Arabidopsis thaliana known to express the target protein

• Negative control: Protein extract from At2g39415 knockout/knockdown lines

• Secondary antibody control: Sample processed with secondary antibody only (no primary antibody)

• Blocking peptide control: Pre-incubation of antibody with its immunizing peptide to verify specificity

• Loading control: Probing for a housekeeping protein (e.g., actin, tubulin) to verify equal loading

These controls help distinguish specific from non-specific signals and validate experimental results, addressing a common concern in antibody research regarding specificity.

How can I validate the specificity of the At2g39415 Antibody?

Antibody specificity is paramount for reliable research outcomes. To validate the At2g39415 Antibody:

• Perform Western blot analysis comparing wild-type and At2g39415 mutant plants

• Conduct immunoprecipitation followed by mass spectrometry analysis to identify all proteins captured by the antibody

• Test cross-reactivity with recombinant proteins of similar structure

• Compare staining patterns in immunohistochemistry with known expression patterns from promoter-reporter studies

• Evaluate antibody specificity across different Arabidopsis ecotypes

This comprehensive validation approach addresses concerns about non-specific binding, which is a documented issue with some commercially available antibodies as seen with AT2 receptor antibodies in other research contexts .

What are the optimal storage and handling conditions for maintaining antibody efficacy?

To preserve At2g39415 Antibody functionality:

Improper storage can lead to reduced binding efficiency, increased background, and experimental variability. Document all freeze-thaw cycles and observe for any changes in performance over time.

How should I optimize the At2g39415 Antibody concentration for Western blotting?

For optimal Western blot results with the At2g39415 Antibody:

• Perform a dilution series experiment (typically 1:500, 1:1000, 1:2000, 1:5000)

• Load consistent protein amounts (20-50μg) across wells

• Transfer proteins to membrane using standard protocols (PVDF often provides better results than nitrocellulose for plant proteins)

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

• Incubate with primary antibody dilutions overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5 minutes each)

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Develop using chemiluminescence and determine optimal dilution based on signal-to-noise ratio

This titration approach helps identify the concentration that provides maximum specific signal with minimal background.

How can I use the At2g39415 Antibody for co-immunoprecipitation to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) with the At2g39415 Antibody enables identification of protein interaction networks:

• Prepare plant lysates under non-denaturing conditions using a gentle lysis buffer:

  • 50mM Tris-HCl (pH 7.5)

  • 150mM NaCl

  • 0.5% NP-40

  • 1mM EDTA

  • Protease and phosphatase inhibitors

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Incubate pre-cleared lysate with At2g39415 Antibody (2-5μg per mg of protein) overnight at 4°C

• Add fresh Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads 5 times with wash buffer (lysis buffer with reduced detergent)

• Elute proteins by boiling in SDS sample buffer

• Analyze by SDS-PAGE followed by:

  • Western blotting for suspected interaction partners

  • Silver staining and mass spectrometry for unbiased partner identification

This approach can reveal physiologically relevant protein complexes and regulatory mechanisms involving the At2g39415 gene product.

What strategies can address epitope masking issues when the At2g39415 protein undergoes post-translational modifications?

Post-translational modifications (PTMs) can interfere with antibody recognition. When working with the At2g39415 Antibody:

• Phosphorylation interference: Treat samples with lambda phosphatase to remove phosphate groups that might mask epitopes

• Glycosylation masking: Use PNGase F or Endo H treatment to remove N-linked glycans

• Conformational epitope access: Optimize denaturation conditions or test native vs. denatured detection

• Multiple antibody approach: Use antibodies recognizing different epitopes of the same protein

• PTM-specific antibodies: Consider using antibodies specifically designed to recognize modified forms

These strategies help ensure comprehensive detection of the target protein regardless of its modification state, providing insights into the protein's regulatory mechanisms.

How can I effectively use the At2g39415 Antibody for chromatin immunoprecipitation (ChIP) experiments?

For researchers investigating DNA-protein interactions involving the At2g39415 gene product:

• Crosslink plant tissue with 1% formaldehyde for 10-15 minutes under vacuum

• Quench with 125mM glycine for 5 minutes

• Extract and sonicate chromatin to fragments of 200-500bp

• Pre-clear chromatin with Protein A/G beads

• Incubate pre-cleared chromatin with At2g39415 Antibody (5-10μg) overnight at 4°C

• Add fresh Protein A/G beads and incubate for 2-4 hours

• Perform sequential washes with increasing stringency buffers

• Reverse crosslinks by heating at 65°C overnight

• Purify DNA and analyze by qPCR or sequencing

ChIP experiments can reveal genomic binding sites and regulatory functions of the At2g39415 protein if it participates in transcriptional regulation or chromatin remodeling.

What are common causes of false positive/negative results with the At2g39415 Antibody and how can they be addressed?

This systematic approach to troubleshooting helps researchers distinguish between technical issues and genuine biological phenomena. Similar methodology has been employed with antibodies in SARS-CoV-2 research, where specificity is critical .

How should I interpret contradictory results between the At2g39415 Antibody and other detection methods?

When facing discrepancies between antibody-based detection and other methods:

• Verify antibody specificity through knockout/knockdown validation

• Consider the epitope location and whether it might be masked in certain contexts

• Evaluate protein expression levels through orthogonal methods:

  • qRT-PCR for mRNA expression

  • Reporter gene fusions for protein localization

  • Mass spectrometry for protein identification and quantification

• Assess if detection thresholds differ between methods

• Consider if post-translational modifications affect detection

• Evaluate if protein-protein interactions obscure epitope accessibility

Method triangulation increases confidence in results and helps identify technique-specific limitations. Recent studies with therapeutic antibodies demonstrate the importance of using multiple validation approaches .

How can I determine if batch-to-batch variation is affecting my At2g39415 Antibody experiments?

Batch variation is a significant concern in antibody research. To monitor and address this issue:

• Maintain a reference sample set tested with the original antibody batch

• Run side-by-side comparisons with old and new antibody batches

• Document key performance metrics:

  • Signal-to-noise ratio

  • Detection threshold

  • Banding pattern in Western blots

  • Localization pattern in immunostaining

• Request lot-specific validation data from suppliers

• Consider preparing a large single batch of antibody for long-term projects

• Implement a standard curve with recombinant protein to normalize across experiments

This approach aligns with best practices observed in biomedical research, where antibody reliability issues have been documented and require systematic assessment .

How can I use the At2g39415 Antibody to investigate protein dynamics during plant stress responses?

To study At2g39415 protein dynamics during stress:

• Design time-course experiments exposing plants to relevant stressors (drought, salt, pathogen, temperature)

• Collect tissue samples at defined intervals (0, 1, 3, 6, 12, 24, 48 hours)

• Process parallel samples for:

  • Protein extraction and Western blotting with At2g39415 Antibody

  • RNA extraction and qRT-PCR for transcript analysis

  • Immunolocalization to track protein redistribution

• Quantify protein levels through densitometry

• Correlate protein abundance with transcriptional changes

• Track post-translational modifications using phospho-specific antibodies if available

• Perform co-immunoprecipitation at key timepoints to identify stress-specific interaction partners

This comprehensive approach provides insights into protein regulation mechanisms during stress adaptation.

What considerations are important when using the At2g39415 Antibody for super-resolution microscopy?

For super-resolution microscopy applications:

• Sample preparation optimization:

  • Use thin tissue sections (≤10μm) or isolated cells

  • Optimize fixation protocols to preserve antigenicity while maintaining structure

  • Evaluate alternative fixation methods (paraformaldehyde vs. methanol vs. glutaraldehyde)

• Antibody considerations:

  • Use highly purified antibody preparations

  • Consider directly conjugated primary antibodies to improve localization precision

  • Validate specificity at the subcellular level using knockout controls

  • Test different antibody concentrations to maximize signal while minimizing background

• Imaging parameters:

  • Select appropriate fluorophores with photostability suitable for super-resolution

  • Optimize imaging buffers to enhance fluorophore performance

  • Include fiducial markers for drift correction

  • Perform multicolor imaging with carefully selected non-overlapping fluorophores

• Data analysis:

  • Apply appropriate image processing algorithms

  • Quantify colocalization with subcellular markers

  • Perform statistical analysis of spatial distribution

These considerations ensure reliable protein localization at nanoscale resolution, providing insights into protein function within cellular compartments.

How can the At2g39415 Antibody be used in combination with CRISPR-Cas9 gene editing to validate gene function?

Integrating antibody detection with CRISPR-Cas9 technology:

• Design and generate CRISPR-Cas9 edited Arabidopsis lines:

  • Complete gene knockout

  • Domain-specific mutations

  • Epitope-tagged versions (ensuring tags don't interfere with antibody binding)

• Validation experiments:

  • Confirm editing through sequencing

  • Verify protein reduction/modification by Western blotting with At2g39415 Antibody

  • Assess subcellular localization changes via immunofluorescence

  • Quantify protein-protein interaction alterations through co-immunoprecipitation

• Phenotypic analysis:

  • Document morphological changes

  • Measure physiological parameters

  • Assess stress responses

  • Analyze developmental timing

• Rescue experiments:

  • Complement edited lines with wild-type or modified gene versions

  • Verify protein expression using the At2g39415 Antibody

  • Correlate protein levels with phenotypic rescue

This combined approach provides robust evidence for gene function and protein activity, similar to validation strategies used in therapeutic antibody research .

What emerging technologies might enhance the utility of the At2g39415 Antibody in plant research?

Several cutting-edge technologies hold promise for expanding At2g39415 Antibody applications:

• Proximity labeling: Conjugating biotin ligases (BioID, TurboID) to antibodies for identifying transient or weak interaction partners

• Single-cell proteomics: Combining the antibody with microfluidics and mass cytometry for cell-specific protein quantification

• Intrabodies: Developing cell-permeable versions for live-cell imaging and protein manipulation

• Nanobodies: Engineering smaller antibody fragments for improved tissue penetration and reduced immunogenicity

• Biolayer interferometry: Using antibodies in label-free, real-time binding assays

• Spatial transcriptomics integration: Combining immunolocalization with spatial gene expression analysis

• Cryo-electron tomography: Using gold-conjugated antibodies for 3D ultrastructural localization

These technologies may overcome current limitations in detecting low-abundance proteins, resolving temporal dynamics, and identifying cell-type-specific functions of the At2g39415 gene product.

How might computational approaches improve At2g39415 Antibody-based research?

Computational methods enhance antibody research through:

• Epitope prediction algorithms: Identifying optimal antibody binding sites

• Molecular dynamics simulations: Predicting antibody-antigen interactions and conformational changes

• Machine learning for image analysis: Automating protein localization and quantification in microscopy data

• Network analysis tools: Interpreting protein-protein interaction data from co-immunoprecipitation

• Integrative multi-omics analysis: Correlating antibody-based proteomics with transcriptomics and metabolomics

• Phylogenetic analysis: Predicting cross-reactivity with related proteins across species

• Quantitative image analysis: Extracting numerical data from immunohistochemistry for statistical comparison

These computational approaches transform qualitative antibody data into quantitative insights while improving experimental design and interpretation.