At2g39518 Antibody

What is At2g39518 and why are antibodies against this protein important for plant research?

At2g39518 is an Arabidopsis thaliana gene locus coding for a protein involved in plant cellular processes. Antibodies against this protein are essential research tools for studying protein localization, expression patterns, and functional characterization in plant biology. The development of specific antibodies enables researchers to track this protein's distribution at subcellular, cellular, and tissue levels, providing critical insights into its biological function and regulatory networks in plant development .

Antibodies targeting Arabidopsis proteins like At2g39518 have become invaluable for integrative systems biology approaches, where understanding protein localization contributes to deciphering protein-protein interactions and regulatory networks. These antibodies facilitate research in multiple experimental contexts including immunohistochemistry, western blotting, and co-immunoprecipitation studies .

How are antibodies against Arabidopsis proteins like At2g39518 typically generated?

Antibodies against Arabidopsis proteins like At2g39518 are typically generated using two main approaches:

The recombinant protein approach is generally more successful, with studies showing that approximately 55% (38 of 70) of antibodies generated using this method could detect their target proteins with high confidence .

What validation methods should be used to confirm At2g39518 antibody specificity?

Validation of At2g39518 antibody specificity should follow a multi-step process to ensure reliability:

For enhanced validation, the antibody should meet stringent criteria based on orthogonal validation or independent antibody validation strategies . When possible, the antibody should be tested against corresponding mutant backgrounds to confirm specificity. A complete absence of signal in the mutant background provides strong evidence for antibody specificity .

What is the recommended protocol for using At2g39518 antibody in immunolocalization studies?

For optimal results in immunolocalization studies with At2g39518 antibody, follow this methodological approach:

• Tissue preparation: Fix Arabidopsis root or shoot tissues in 4% paraformaldehyde in PBS for 1 hour at room temperature. After fixation, embed tissues in paraffin or prepare for whole-mount immunolocalization.

• Sectioning and permeabilization: For paraffin sections, cut 5-10 μm sections. For whole-mount, permeabilize with 0.1-0.5% Triton X-100 in PBS for 15 minutes. Block non-specific binding with 3% BSA in PBS for 1 hour.

• Primary antibody incubation: Dilute the At2g39518 antibody at 1:100 to 1:500 (determine optimal dilution empirically) in blocking solution. Incubate overnight at 4°C.

• Washing and secondary antibody: Wash 3 times with PBS containing 0.1% Tween-20. Incubate with fluorophore-conjugated secondary antibody (1:500 dilution) for 2 hours at room temperature.

• Final washes and mounting: Wash 3 times with PBS containing 0.1% Tween-20. Mount in anti-fade mounting medium containing DAPI for nuclear counterstaining.

• Controls: Always include negative controls (secondary antibody only, pre-immune serum) and, if available, tissues from At2g39518 knockout/mutant plants .

The success rate of immunocytochemistry-grade antibodies for Arabidopsis proteins is approximately 30% (22 of 70 antibodies), so optimization may be necessary for specific experimental conditions .

What are the key considerations for Western blot analysis using At2g39518 antibody?

When performing Western blot analysis with At2g39518 antibody, consider these critical methodological factors:

• Protein extraction: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail. Plant tissues often contain interfering compounds, so include 2% PVPP and 5 mM DTT to prevent oxidation.

• Sample preparation: Heat samples at 70°C for 10 minutes (rather than boiling) to reduce protein aggregation. Load 10-30 μg total protein per lane.

• Gel electrophoresis and transfer: Use 10-12% SDS-PAGE gels and transfer to PVDF membranes (preferred over nitrocellulose for plant proteins).

• Blocking and antibody incubation: Block with 5% non-fat dry milk in TBST. Use At2g39518 antibody at 1:1000 to 1:5000 dilution (optimize empirically). Extend primary antibody incubation to overnight at 4°C for better results.

• Detection: Use enhanced chemiluminescence with exposure times of 30 seconds to 5 minutes. For low abundance proteins, consider using more sensitive detection systems or signal enhancement methods.

• Validation: Always include positive controls (tissues known to express At2g39518) and negative controls (At2g39518 mutant/knockdown tissues if available) .

The expected molecular weight of the At2g39518 protein should be determined based on its amino acid sequence, and the observed band size should match this prediction for proper identification.

How can I improve the sensitivity of At2g39518 antibody detection in low-expressing tissues?

For detecting At2g39518 protein in tissues with low expression levels, implement these methodological enhancements:

• Antibody affinity purification: Affinity purification of antibodies can significantly improve detection sensitivity. Studies have shown that affinity purification "massively improved the detection rate" for plant protein antibodies .

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) for immunohistochemistry, which can increase sensitivity 10-100 fold

  • Employ biotin-streptavidin amplification systems for Western blots

  • Consider using quantum dot-conjugated secondary antibodies for enhanced fluorescence detection

• Sample enrichment techniques:

  • Perform subcellular fractionation to concentrate the protein of interest

  • Use immunoprecipitation prior to Western blotting

  • Enrich for membrane proteins if At2g39518 is membrane-associated

• Reducing background:

  • Extend blocking times to 2-3 hours

  • Include 0.1-0.3% Tween-20 in antibody dilution buffers

  • Use more stringent washing conditions (higher salt concentration)

  • Pre-absorb antibodies with plant extracts from At2g39518 knockout plants

• Detection optimization:

  • Use highly sensitive ECL substrates for Western blots

  • Extend exposure times for imaging

  • Employ digital imaging systems with cooling capabilities for reduced background .

Implementing these techniques can improve detection sensitivity by 2-10 fold, making it possible to detect proteins even when expressed at very low levels.

How can computational modeling improve At2g39518 antibody development and specificity?

Computational modeling approaches can significantly enhance At2g39518 antibody development through these methodological strategies:

• Epitope prediction and optimization:

  • Implement Rosetta-based approaches to model the protein-antibody binding interface

  • Use dTERMen, an informatics approach, to generate predictions for mutations that improve binding

  • Apply sliding window analysis to identify epitopes with <40% sequence similarity to other Arabidopsis proteins

• Structural modeling for discontinuous epitopes:

  • Model tertiary structure to identify discontinuous epitopes that involve distant subsequences brought together by protein folding

  • Predict conformational stability of epitopes to ensure they maintain native structure

• Affinity maturation pipelines:

  • Integrate computational modeling with experimental library screening to increase antibody affinity

  • Create phage display libraries of scFvs incorporating predicted mutations

  • Screen libraries for binding affinity to the recombinant antigen

  • Incorporate favorable mutations into final antibody constructs

This integrated approach can improve antibody affinity by up to 60-fold (KD improvement from 0.63 nM to 0.01 nM has been demonstrated in similar applications) . For plant proteins like At2g39518, computational modeling should account for potential post-translational modifications specific to plant systems.

What strategies can address cross-reactivity issues with At2g39518 antibody in multi-gene family proteins?

When At2g39518 belongs to a multi-gene family with high sequence similarity among members, these specialized approaches can minimize cross-reactivity:

• Unique region targeting:

  • Perform comprehensive sequence alignment of all family members

  • Identify regions with <40% sequence similarity using sliding window analysis

  • Target antibody development to these unique regions, even if they are smaller than ideal (~100 amino acids)

• Validation in genetic backgrounds:

  • Test antibody specificity in plants with mutations in At2g39518

  • Examine cross-reactivity with closely related family members using overexpression lines

  • Use RNA interference or CRISPR knockdown lines to validate specificity

• Epitope engineering:

  • Introduce subtle mutations in recombinant proteins to enhance uniqueness

  • Design peptides spanning junctions between domains that are less conserved

  • Generate competitive peptides for blocking cross-reactive epitopes

• Advanced purification strategies:

  • Perform negative selection against related family members

  • Use sequential affinity purification to remove cross-reactive antibodies

  • Consider subtractive immunization protocols

When it is impossible to obtain a unique sequence of sufficient size, researchers should consider developing "family-specific" antibodies and then using genetic or biochemical approaches to distinguish between family members in experimental contexts .

How can At2g39518 antibody be utilized in protein interaction and regulatory network studies?

At2g39518 antibody can be leveraged for sophisticated protein interaction and regulatory network analyses through these methodological applications:

• Co-immunoprecipitation (Co-IP) studies:

  • Use At2g39518 antibody conjugated to magnetic or agarose beads

  • Isolate protein complexes from plant tissues under native conditions

  • Identify interaction partners using mass spectrometry

  • Validate interactions with reciprocal Co-IP using antibodies against putative partners

• Chromatin immunoprecipitation (ChIP) applications:

  • If At2g39518 protein has DNA-binding properties, perform ChIP to identify genomic binding sites

  • Combine with sequencing (ChIP-seq) for genome-wide binding profile

  • Integrate with transcriptome data to identify regulated genes

• Proximity-dependent labeling:

  • Engineer fusion proteins combining At2g39518 with BioID or APEX2

  • Use antibodies to validate expression and localization of fusion proteins

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Dynamic protein localization:

  • Track protein redistribution during different developmental stages or stress responses

  • Perform co-localization studies with markers for different organelles

  • Quantify protein levels in different subcellular compartments

• Protein modification analysis:

  • Combine immunoprecipitation with phospho-specific antibodies to study phosphorylation

  • Use the antibody to pull down At2g39518 and analyze post-translational modifications

  • Study how modifications affect protein interactions and function

These approaches enable researchers to move beyond simple localization studies to understand the functional role of At2g39518 in plant cellular systems, particularly in protein-protein interactions and regulatory networks important for plant development and response to environmental conditions.

How can I troubleshoot inconsistent results between At2g39518 antibody detection and RNA expression data?

When facing discrepancies between antibody detection and RNA expression data for At2g39518, implement this systematic troubleshooting approach:

• Assess antibody reliability score:

  • Evaluate the antibody against established reliability criteria (Enhanced, Supported, Approved, or Uncertain)

  • For high reliability ("Enhanced" score), antibodies should meet stringent validation criteria including orthogonal validation or independent antibody validation

• Analyze potential biological explanations:

  • Post-transcriptional regulation may cause protein levels to differ from RNA levels

  • Protein turnover rates can affect steady-state protein abundance

  • Tissue-specific translational control may explain localized discrepancies

• Examine technical factors:

  • RNA detection methods may have different sensitivity than protein detection

  • Protein extraction efficiency can vary between tissues

  • Antibody accessibility to epitopes may be affected by protein folding or interactions

• Apply RNA similarity scoring:

  RNA Similarity Score	Definition	Interpretation
  High consistency	Strong correlation between RNA and protein	Reliable antibody performance
  Medium consistency	Moderate correlation with some variation	Generally reliable with caveats
  Low consistency	Poor correlation with significant discrepancies	Requires additional validation
  Very low consistency	No correlation between RNA and protein data	May indicate antibody problems
  Cannot be evaluated	Insufficient data for comparison	Additional tests needed

• Validation strategies:

  • Validate with independent techniques (e.g., tagged protein expression)

  • Test in multiple biological replicates and conditions

  • Consider temporal dynamics of RNA vs. protein expression

A comprehensive analysis combining these approaches can help determine whether discrepancies represent technical issues with the antibody or interesting biological phenomena worth further investigation.

What statistical approaches are recommended for quantifying At2g39518 protein levels in comparative studies?

For rigorous quantification of At2g39518 protein levels in comparative studies, implement these statistical and methodological approaches:

• Experimental design considerations:

  • Employ a completely randomized design or randomized block design

  • Include biological replicates (minimum n=3, preferably n=5 or greater)

  • Process control and experimental samples simultaneously to minimize batch effects

• Quantification methods:

  • For Western blots: Use densitometry with appropriate normalization to loading controls

  • For immunohistochemistry: Implement fluorescence intensity measurement with background subtraction

  • For flow cytometry: Calculate mean fluorescence intensity with appropriate gating strategies

• Statistical analysis framework:

  Analysis Type	Application	Statistical Method
  Two-group comparison	Comparing mutant vs. WT	Student's t-test or Mann-Whitney U test
  Multi-group comparison	Multiple treatments or genotypes	One-way ANOVA with appropriate post-hoc tests
  Time-course or developmental series	Expression changes over time	Repeated measures ANOVA or mixed-effects models
  Correlation analysis	Relationship to physiological parameters	Pearson's or Spearman's correlation coefficient

• Data normalization strategies:

  • Normalize to stable reference proteins (not housekeeping genes that may vary)

  • Consider global normalization methods for large-scale studies

  • Implement variance stabilizing transformations for heteroscedastic data

• Reporting standards:

  • Report both raw and normalized data

  • Include measures of variability (standard deviation or standard error)

  • Provide clear descriptions of statistical tests and significance thresholds

  • Report effect sizes alongside p-values

Following these rigorous quantification approaches ensures that comparative studies of At2g39518 protein expression yield reliable, reproducible, and statistically sound results.

How can I distinguish between specific and non-specific binding in At2g39518 antibody applications?

To effectively differentiate between specific and non-specific binding in At2g39518 antibody applications, implement this comprehensive validation framework:

• Controls for specificity determination:

  • Genetic controls: Test in At2g39518 knockout/mutant tissues (gold standard)

  • Peptide competition: Pre-incubate antibody with immunizing peptide/protein

  • Isotype controls: Use matched isotype antibody from non-immunized animals

  • Secondary-only controls: Omit primary antibody to detect non-specific secondary binding

• Signal evaluation criteria:

  Observation	Interpretation	Action Required
  Single band of expected size	Likely specific binding	Proceed with experiments
  Multiple bands including expected size	Partial specificity	Consider affinity purification
  Bands in knockout tissue	Non-specific binding	Optimize conditions or reject antibody
  Background staining in all tissues	Poor signal-to-noise ratio	Modify blocking or antibody concentration

• Optimization approaches:

  • Test a range of antibody dilutions (1:100 to 1:10,000)

  • Modify blocking agents (BSA, non-fat milk, normal serum)

  • Adjust detergent concentration in wash buffers

  • Implement antigen retrieval methods for fixed tissues

• Advanced validation methods:

  • Use orthogonal methods like mass spectrometry to confirm detected proteins

  • Apply independent antibodies raised against different epitopes

  • Validate with epitope-tagged versions of At2g39518 protein

For immunohistochemistry applications, the pattern of staining should be consistent with the expected subcellular localization and tissue distribution based on transcript data. The signal should be absent or significantly reduced in knockout/mutant tissues, providing strong evidence for antibody specificity .

How can nanobody technology improve At2g39518 protein research compared to conventional antibodies?

Nanobody technology offers several methodological advantages for At2g39518 protein research compared to conventional antibodies:

• Enhanced epitope accessibility:

  • Nanobodies (15 kDa) are significantly smaller than conventional antibodies (150 kDa)

  • Their small size allows access to epitopes in protein complexes or membrane proteins that may be inaccessible to conventional antibodies

  • This is particularly valuable for studying structural proteins or those in crowded cellular environments

• Superior specificity for active sites:

  • Nanobodies can effectively target active sites of proteins

  • They can potentially interfere with protein function by binding to functional domains

  • This property enables not just detection but functional modulation of target proteins

• Improved intracellular applications:

  • Nanobodies can be expressed intracellularly as "intrabodies"

  • They maintain stability in the reducing intracellular environment

  • This allows real-time tracking of native At2g39518 in living cells

• Research applications comparison:

  Application	Conventional Antibodies	Nanobodies	Advantage
  Western blotting	Standard practice	Comparable performance	Similar results
  Immunoprecipitation	Requires secondary capture	Direct coupling possible	Cleaner results
  Live cell imaging	Limited to surface proteins	Intracellular expression	Dynamic studies
  Crystallography	Rare success	Frequent crystallization chaperones	Structural insights
  Therapeutic potential	Clinical use established	Clinical trials underway	Less immunogenicity

• Production advantages:

  • Nanobodies can be produced in bacterial systems

  • They show high thermostability and pH resistance

  • They maintain functionality in varying buffer conditions

The ability of nanobodies to target At2g39518 could provide new insights into protein function while offering research tools with potentially superior specificity and versatility compared to conventional antibodies.

What are the latest approaches for multiplexed detection of At2g39518 alongside other proteins of interest?

Recent technological advances enable sophisticated multiplexed detection of At2g39518 alongside other proteins through these methodological approaches:

• Multiplex immunofluorescence techniques:

  • Sequential multiplexing: Sequential staining and stripping/quenching approaches

  • Spectral unmixing: Using spectrally overlapping fluorophores with computational separation

  • DNA-barcoded antibodies: Antibodies tagged with unique DNA sequences for multiplexed detection

• Mass cytometry (CyTOF) adaptations for plant tissues:

  • Metal-tagged antibodies enable simultaneous detection of >40 proteins

  • Sample preparation protocols adapted for plant cell walls

  • Computational analysis approaches for high-dimensional data interpretation

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving At2g39518

  • Only generate signal when two target proteins are within 40 nm

  • Can be multiplexed using different oligonucleotide pairs

• Multiplexed Western blotting approaches:

  Method	Principle	Protein Capacity	Advantages
  Fluorescent multiplex	Different fluorophores	4-5 proteins	No stripping required
  Sequential ECL	Strip and reprobe	Unlimited (sequential)	Works with existing antibodies
  Capillary immunoassay	Size separation in capillaries	12+ proteins	Minimal sample requirement

• Spatial transcriptomics integration:

  • Combine antibody detection with in situ mRNA detection

  • Correlate protein localization with gene expression patterns

  • Create comprehensive spatial maps of protein-RNA relationships

These multiplexed approaches enable researchers to study At2g39518 in the context of its interacting partners and signaling networks, providing a systems-level understanding of its function in plant biology.

How will enhanced antibody validation standards impact the reliability of At2g39518 antibody research?

The implementation of enhanced validation standards for At2g39518 antibodies will transform research reliability through these systematic improvements:

• Multi-tier validation framework:

  • Reliability scoring system categorizing antibodies as "Enhanced," "Supported," "Approved," or "Uncertain"

  • Enhanced validation requiring stringent criteria based on orthogonal validation or independent antibody validation

  • Transparency in reporting validation methods and results

• Validation criteria integration:

  Validation Level	Required Evidence	Impact on Research
  Enhanced	Orthogonal validation or independent antibody validation	Highest confidence for critical studies
  Supported	RNA consistency and literature consistency	Suitable for exploratory research
  Approved	Partial RNA consistency or literature support	Requires additional controls
  Uncertain	Inconsistent with RNA or literature	Should be used with extreme caution

• Methodology standardization benefits:

  • Reduction in contradictory research findings

  • Improved reproducibility across laboratories

  • Enhanced ability to compare results between studies

  • More efficient resource utilization by preventing use of unreliable reagents

• Implementation in repository systems:

  • Integration with antibody repositories like Nottingham Arabidopsis Stock Centre

  • Standardized reporting of validation data

  • Community feedback mechanisms to continuously update reliability assessments

• Impact on discovery of poorly characterized proteins:

  • Enhanced validation has already uncovered 56 proteins corresponding to the group of "missing proteins"

  • Similar approaches could validate At2g39518 if it is currently poorly characterized

  • Rigorous validation could lead to discovery of novel functions

The adoption of these enhanced validation standards will significantly increase confidence in At2g39518 antibody research, leading to more reproducible results and accelerating scientific discovery in plant biology.