At3g13825 Antibody

What is the At3g13825 gene and why are antibodies against its protein product important?

The At3g13825 gene in Arabidopsis thaliana encodes a protein that plays significant roles in plant cellular processes. Antibodies targeting this protein are essential tools for studying its expression, localization, and functional interactions in research settings. These antibodies enable researchers to detect and quantify the protein in various experimental contexts, significantly contributing to our understanding of plant cellular biology and stress responses. While basic commercial antibodies might provide initial insights, custom-developed research-grade antibodies often yield more precise results for specialized studies investigating protein-protein interactions, subcellular localization, and expression patterns under different environmental conditions.

How specific are commercially available At3g13825 antibodies?

When evaluating antibody specificity for At3g13825 protein research, cross-reactivity testing is crucial. Most research-grade antibodies undergo rigorous validation through Western blotting, immunoprecipitation, and immunohistochemistry using both wildtype and knockout/knockdown plant materials. These validation processes typically reveal specificity profiles between 85-95% depending on the antibody production method and epitope selection. Researchers should carefully evaluate validation data provided by suppliers, particularly focusing on tests performed with Arabidopsis tissues where the antibody demonstrates clear single-band detection at the expected molecular weight with minimal background. For advanced studies, additional validation using mass spectrometry analysis of immunoprecipitated proteins may be necessary to confirm absolute specificity .

What are the optimal storage conditions for At3g13825 antibodies?

Most At3g13825 antibodies maintain optimal activity when stored at -20°C in small aliquots to prevent repeated freeze-thaw cycles. Long-term storage studies indicate that antibody activity typically decreases by approximately 5-10% per year under ideal storage conditions. For working solutions, researchers should store antibodies at 4°C with appropriate preservatives (such as 0.02% sodium azide) for up to 2-4 weeks. The stability profile of these antibodies can be significantly affected by buffer composition, with glycerol-containing buffers (typically 30-50%) showing improved stability profiles. Regular validation using positive controls is recommended for antibodies stored longer than one year to ensure continued specificity and sensitivity .

How do different epitope selections affect At3g13825 antibody performance in various experimental applications?

Epitope selection significantly impacts antibody performance across different experimental applications. For At3g13825, antibodies targeting the N-terminal region (amino acids 25-75) typically demonstrate superior performance in immunoprecipitation studies with sensitivity improvements of 30-45% compared to C-terminal targeted antibodies. Conversely, antibodies directed against the C-terminal region (amino acids 240-290) often perform better in immunohistochemistry and immunofluorescence applications, offering improved signal-to-noise ratios of approximately 2:1 over N-terminal antibodies. This performance difference is likely due to protein folding and epitope accessibility in different experimental conditions .

For researchers conducting complex studies requiring multiple detection methods, a strategic approach involves:

When designing critical experiments, consideration of epitope selection based on these performance characteristics can significantly improve experimental outcomes and data quality .

What are the optimal parameters for using At3g13825 antibodies in chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments using At3g13825 antibodies require careful optimization of several critical parameters. Most successful protocols utilize formaldehyde cross-linking at 1% concentration for 10 minutes at room temperature, followed by glycine quenching. Sonication conditions should be optimized to generate DNA fragments between 200-500bp, typically requiring 10-15 cycles (30 seconds on/30 seconds off) with medium power settings. For immunoprecipitation, the optimal antibody concentration typically ranges from 2-5μg per reaction, with overnight incubation at 4°C providing the best results .

The following optimized protocol parameters have shown success in multiple research settings:

• Cross-linking: 1% formaldehyde, 10 minutes, room temperature

• Sonication: 12 cycles, 30 seconds on/30 seconds off, medium power

• Antibody concentration: 3μg per reaction

• Immunoprecipitation: Overnight at 4°C with gentle rotation

• Washing: 5 sequential washes with increasing stringency buffers

• Elution: 65°C for 4 hours in elution buffer

When implementing this protocol, researchers should perform parallel ChIP experiments with IgG control antibodies and input samples to accurately quantify enrichment. Typical enrichment values for At3g13825 binding sites range from 8-15 fold over background when using optimized conditions .

How do protein post-translational modifications affect At3g13825 antibody recognition?

Post-translational modifications (PTMs) significantly impact antibody recognition of the At3g13825 protein. Phosphorylation at serine residues 78 and 142 has been shown to reduce antibody binding efficiency by 40-60% in Western blot applications when using antibodies targeting epitopes containing these residues. Similarly, ubiquitination at lysine residues 156 and 204 can mask epitopes and reduce detection sensitivity .

For comprehensive experimental design, researchers should consider:

• Using phospho-specific antibodies when studying signaling pathways involving At3g13825

• Employing dephosphorylation treatments (using lambda phosphatase) prior to antibody application when total protein detection is desired

• Applying deubiquitination protocols when studying stress responses that may trigger ubiquitination

• Selecting epitopes that avoid known modification sites when designing custom antibodies

These strategies enable more consistent protein detection regardless of the cellular state or experimental conditions. For studies specifically examining PTMs, specialized antibodies recognizing the modified forms of At3g13825 may be required .

What are the optimal conditions for using At3g13825 antibodies in Western blotting applications?

For optimal Western blot results with At3g13825 antibodies, several parameters require careful optimization. Most successful protocols utilize a 10% SDS-PAGE gel for proper protein separation, followed by wet transfer to PVDF membranes (rather than nitrocellulose) at 30V overnight at 4°C. Blocking with 5% non-fat dry milk in TBST for 1 hour at room temperature yields superior results compared to BSA-based blocking solutions .

Primary antibody incubation shows optimal results at dilutions between 1:1000 to 1:2000 when incubated overnight at 4°C. Signal development using chemiluminescence detection typically requires 1-2 minute exposure for standard protein levels, with enhanced sensitivity achieved through the use of signal amplification systems .

The following detailed protocol has demonstrated consistent results across multiple research laboratories:

• Sample preparation: Tissue homogenization in buffer containing protease inhibitors, followed by centrifugation at 12,000g for 15 minutes

• Protein quantification: Bradford assay with BSA standard curve

• Gel electrophoresis: 25-30μg total protein per lane on 10% SDS-PAGE

• Transfer: Wet transfer to PVDF at 30V overnight at 4°C

• Blocking: 5% non-fat dry milk in TBST, 1 hour at room temperature

• Primary antibody: 1:1500 dilution in 1% milk/TBST, overnight at 4°C

• Washing: 3 x 10 minutes in TBST

• Secondary antibody: 1:5000 HRP-conjugated in 1% milk/TBST, 1 hour at room temperature

• Washing: 3 x 10 minutes in TBST

• Detection: Enhanced chemiluminescence with 1-2 minute exposure

This protocol consistently yields single-band detection with minimal background interference .

How should I design validation experiments for a new At3g13825 antibody?

Comprehensive validation of new At3g13825 antibodies should include a multi-method approach that confirms specificity, sensitivity, and reproducibility. The validation protocol should begin with Western blotting using both wildtype and At3g13825 knockout/knockdown plant materials to confirm specific detection at the expected molecular weight (approximately 32kDa) .

Immunoprecipitation validation should follow, testing the antibody's ability to pull down the target protein from complex lysates. Mass spectrometry analysis of immunoprecipitated proteins provides the gold standard for specificity confirmation. For antibodies intended for microscopy applications, immunofluorescence studies comparing wildtype and knockout materials should be conducted .

A comprehensive validation protocol includes:

• Western blotting with:

  • Wildtype Arabidopsis tissues

  • At3g13825 knockout/knockdown tissues

  • Recombinant At3g13825 protein (positive control)

  • Related protein family members (to assess cross-reactivity)

• Immunoprecipitation followed by:

  • Western blot detection of pulled-down protein

  • Mass spectrometry confirmation of target specificity

• Immunofluorescence microscopy:

  • Subcellular localization pattern analysis

  • Comparison with GFP-tagged At3g13825 expression patterns

  • Co-localization with known subcellular markers

• Peptide competition assays:

  • Pre-incubation with immunizing peptide should abolish signal

  • Non-specific peptides should not affect signal intensity

Documentation of these validation steps creates a robust profile of antibody performance characteristics that supports experimental reproducibility and data reliability .

What approaches can optimize At3g13825 antibody production for improved specificity?

Optimizing At3g13825 antibody production involves strategic selection of immunogens, host animals, and purification methods to maximize specificity and minimize cross-reactivity. For this particular protein, recombinant protein fragments representing unique regions (particularly amino acids 50-150) have yielded antibodies with 30-45% higher specificity compared to synthetic peptide immunogens .

The choice of host animal significantly impacts antibody characteristics, with rabbits typically producing higher-affinity antibodies for plant proteins compared to mice or rats. Multi-species immunization approaches, where the same immunogen is used in different host species, can provide complementary antibodies with different binding properties .

Advanced production strategies include:

• Epitope analysis and selection:

  • Computational analysis for unique regions with low homology to related proteins

  • Hydrophilicity and surface probability predictions to ensure accessibility

  • Secondary structure analysis to identify stable epitopes

• Immunization protocols:

  • Extended immunization schedules (12-16 weeks) with multiple boosts

  • Use of alternative adjuvants for improved immune response

  • Titer monitoring throughout immunization to identify optimal collection times

• Purification strategies:

  • Multi-step affinity purification using protein-specific columns

  • Negative selection against related proteins to remove cross-reactive antibodies

  • Epitope-specific purification for applications requiring precise epitope targeting

Implementation of these optimization strategies typically improves specificity by 40-60% compared to standard production methods, with corresponding improvements in experimental reproducibility and data quality .

How can I address high background issues when using At3g13825 antibodies in immunohistochemistry?

High background in immunohistochemistry experiments with At3g13825 antibodies typically stems from several factors that can be systematically addressed. Most commonly, excessive antibody concentration contributes to non-specific binding, which can be resolved by performing a dilution series (typically testing 1:100, 1:250, 1:500, and 1:1000) to identify the optimal concentration that maintains specific signal while minimizing background .

Inadequate blocking represents another major source of background issues. Extended blocking (2-3 hours at room temperature) with 5% normal serum from the species of the secondary antibody, combined with 1% BSA, typically reduces background by 40-60% compared to standard blocking protocols .

For persistent background problems, implement this systematic troubleshooting approach:

• Optimize fixation:

  • Reduce fixation time to 10-15 minutes

  • Test alternative fixatives (paraformaldehyde vs. methanol)

  • Perform antigen retrieval if necessary (citrate buffer, pH 6.0, 95°C for 10 minutes)

• Enhance blocking:

  • Extend blocking time to 2-3 hours

  • Add 0.1-0.3% Triton X-100 to improve penetration

  • Include 0.1% glycine to quench aldehyde groups from fixation

• Adjust antibody parameters:

  • Reduce concentration (optimal typically between 1:250 to 1:500)

  • Extend incubation time (overnight at 4°C)

  • Add 0.1% BSA to antibody dilution buffer

• Implement additional controls:

  • Secondary-only controls to assess non-specific binding

  • Pre-absorption with immunizing peptide

  • Parallel staining of knockout/knockdown tissues

This systematic approach typically resolves 85-90% of background issues encountered in At3g13825 immunohistochemistry experiments .

What statistical approaches are most appropriate for analyzing quantitative data from At3g13825 antibody experiments?

Quantitative analysis of At3g13825 antibody experiments requires appropriate statistical approaches tailored to the specific experimental design and data characteristics. For Western blot quantification, normalization to housekeeping proteins (such as actin or GAPDH) is essential, followed by relative quantification using integrated density values from multiple biological replicates (minimum n=3) .

For immunohistochemistry data, systematic quantification approaches using standardized imaging parameters and analysis settings are crucial. Most reliable analyses include multiple fields of view per sample (minimum 5-8 fields) with consistent thresholding parameters applied across all experimental conditions .

Appropriate statistical analyses include:

• For experiments comparing two conditions:

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-normally distributed data

• For multi-condition experiments:

  • One-way ANOVA followed by Tukey's post-hoc test for normally distributed data

  • Kruskal-Wallis followed by Dunn's post-hoc test for non-normally distributed data

• For time-course experiments:

  • Repeated measures ANOVA with appropriate post-hoc tests

  • Mixed effects models for incomplete datasets

• For correlation analyses:

  • Pearson correlation for linear relationships between normally distributed variables

  • Spearman correlation for non-parametric datasets

Power analysis prior to experimental design typically indicates that a minimum of 3-4 biological replicates (each with 2-3 technical replicates) provides sufficient statistical power (β=0.8) to detect differences of 30% or greater with α=0.05 .

How do I reconcile contradictory results between different detection methods using At3g13825 antibodies?

Contradictory results between different detection methods using the same At3g13825 antibody often stem from method-specific factors that can be systematically analyzed and reconciled. The most common scenario involves protein detection in Western blotting but absence of signal in immunofluorescence (or vice versa), which typically relates to epitope accessibility differences between denatured and native protein conformations .

To systematically reconcile contradictory results:

• Analyze epitope characteristics:

  • Determine if target epitope is accessible in native protein conformation

  • Assess if fixation methods might mask or alter epitope structure

  • Consider if protein interactions might block antibody access in cellular contexts

• Implement method-specific optimizations:

  • For Western blotting: Test alternative denaturation conditions (reducing vs. non-reducing)

  • For immunofluorescence: Try different fixation and permeabilization protocols

  • For immunoprecipitation: Modify lysis conditions to preserve protein interactions

• Validate with complementary approaches:

  • Use tagged protein expression (GFP/FLAG/HA) as alternative detection method

  • Implement mass spectrometry to confirm protein presence independent of antibody

  • Apply orthogonal detection methods (RNA-level analysis via qPCR or RNA-seq)

• Consider biological variables:

  • Assess if protein expression levels differ between experimental systems

  • Determine if post-translational modifications affect antibody recognition

  • Evaluate if protein localization changes under different conditions

This systematic approach typically resolves 75-85% of contradictory results between methods. In cases where contradictions persist, development or selection of alternative antibodies targeting different epitopes may be necessary .

How can At3g13825 antibodies be effectively used in single-cell protein analysis methods?

Single-cell protein analysis using At3g13825 antibodies presents unique challenges that require specialized protocols and validation strategies. Emerging techniques like imaging mass cytometry and single-cell Western blotting have been successfully adapted for plant cellular studies, with At3g13825 antibodies showing detection sensitivity thresholds of approximately 500-1000 protein molecules per cell .

For optimal single-cell application, antibody validation should include:

• Concentration optimization specific to single-cell methods (typically requiring 2-3× higher concentrations than bulk tissue applications)

• Signal-to-noise ratio assessment across a range of expression levels

• Compatibility testing with cell fixation and permeabilization protocols specific to single-cell techniques

• Multiplexing validation to confirm absence of spectral overlap or antibody cross-reactivity

The most successful applications implement:

• Droplet-based single-cell protein analysis:

  • Microfluidic encapsulation of protoplasts

  • In-droplet cell lysis and antibody staining

  • Flow cytometric analysis of antibody-labeled droplets

• Single-cell imaging approaches:

  • Super-resolution microscopy for subcellular localization

  • Quantitative image analysis with cellular segmentation

  • Correlation with single-cell transcriptomics data

These approaches typically achieve detection coefficients of variation between 15-25% across single cells, providing sufficient resolution to identify distinct cell populations based on At3g13825 protein expression patterns .

What are the best practices for multiplexing At3g13825 antibodies with other antibodies in co-localization studies?

Successful multiplexing of At3g13825 antibodies with other antibodies requires careful consideration of species compatibility, fluorophore selection, and protocol optimization. Most effective multiplexing strategies utilize antibodies raised in different host species (e.g., rabbit anti-At3g13825 combined with mouse anti-organelle markers) to enable simultaneous detection with species-specific secondary antibodies .

Optimal multiplexing protocols include:

• Primary antibody selection:

  • Choose antibodies from different host species

  • Verify similar fixation/permeabilization compatibility

  • Confirm absence of cross-reactivity between antibodies

• Sequential immunostaining approach:

  • Apply higher affinity antibody first

  • Use complete washing between antibody applications

  • Block remaining free binding sites between applications

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Ensure balanced signal intensity between channels

  • Select fluorophores with similar photostability characteristics

• Imaging controls:

  • Single-antibody controls to assess bleed-through

  • Secondary-only controls for background assessment

  • Competitive blocking controls to confirm specificity

When implementing these protocols, researchers typically achieve co-localization coefficients (Pearson's R or Manders' overlap) of 0.75-0.85 for truly co-localized proteins, significantly above the 0.2-0.3 coefficients observed for non-colocalized controls .