At3g51171 Antibody

What is the At3g51171 antibody and what organism does it target?

The At3g51171 antibody is a polyclonal antibody raised in rabbits specifically designed to recognize and bind to the At3g51171 protein from Arabidopsis thaliana (Mouse-ear cress). It is generated using recombinant Arabidopsis thaliana At3g51171 protein as the immunogen . This antibody is primarily designed for plant molecular biology research applications focusing on protein detection and characterization in the model plant organism Arabidopsis thaliana.

What are the validated applications for At3g51171 antibody?

The At3g51171 antibody has been validated for several experimental applications, with the primary validated uses being:

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of the target protein

• Western Blotting (WB) - For identification of the target antigen in protein extracts

These applications have been specifically tested to ensure proper identification of the antigen. The antibody's high specificity makes it suitable for fundamental protein characterization studies in Arabidopsis research.

What are the optimal storage conditions for At3g51171 antibody?

For maximum stability and retention of activity, the At3g51171 antibody should be stored at -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can compromise antibody performance and lead to degradation . The antibody is supplied in liquid form in a storage buffer containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 (preservative)

This formulation helps maintain stability during long-term storage. For working aliquots that will be used frequently, maintaining small volumes at 4°C for up to one month can reduce freeze-thaw damage.

How should researchers optimize the working dilution of At3g51171 antibody for Western blotting?

Optimization of the At3g51171 antibody working dilution for Western blotting requires a systematic approach:

• Begin with a dilution series titration experiment using a range of concentrations (typically from 1:500 to 1:5000)

• Use standardized protein loading (20-30 μg total protein per lane)

• Include positive and negative controls

As an antigen-affinity purified polyclonal antibody, the optimal working dilution is typically in the range of 1.7-15 μg/mL, which is generally lower than for monoclonal antibodies (5-25 μg/mL) . The table below provides a starting framework for dilution optimization:

When comparing different dilutions, maintain consistent blocking reagents, incubation times and temperatures to properly evaluate the signal-to-noise ratio .

What controls should be included when validating At3g51171 antibody specificity?

Rigorous validation of At3g51171 antibody specificity requires several critical controls:

• Positive control: Extract from wild-type Arabidopsis tissue known to express At3g51171

• Negative control: Extract from At3g51171 knockout mutant line or tissue lacking At3g51171 expression

• Preabsorption control: Preincubate antibody with excess purified antigen before immunostaining

• Secondary antibody-only control: Omit primary antibody incubation step

• Cross-reactivity assessment: Test against related Arabidopsis proteins or tissues from other plant species

These controls help distinguish specific signal from background and non-specific binding. Protein microarray technology can be particularly valuable for specificity testing - studies have shown that properly validated antibodies should recognize their target without cross-reacting with other proteins on Arabidopsis protein chips containing 95+ different proteins .

How can researchers minimize background when using At3g51171 antibody in immunological applications?

Background reduction when using At3g51171 antibody requires optimization of several experimental parameters:

• Blocking optimization: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) to determine which gives lowest background for your specific sample

• Antibody concentration: Use the lowest effective concentration that still provides clear specific signal

• Extended wash steps: Increase number and duration of wash steps (at least 3×10 minutes with gentle agitation)

• Detergent adjustment: Fine-tune Tween-20 concentration in wash buffers (0.05-0.1%)

• Sample preparation: Ensure complete protein denaturation for Western blots

• Cross-adsorption: Pre-adsorb the antibody with plant protein extracts from negative control tissue

In immunohistochemistry applications, endogenous peroxidase or phosphatase activity in plant tissues should be blocked before antibody incubation . When using fluorescent detection systems, include an autofluorescence control to distinguish true signal from plant tissue autofluorescence.

How can At3g51171 antibody be integrated into protein microarray studies?

At3g51171 antibody can be effectively utilized in protein microarray studies through the following methodological approach:

• Array preparation: At3g51171 protein can be spotted alongside other Arabidopsis proteins on nitrocellulose-coated FAST slides or polyacrylamide (PAA) slides

• Detection sensitivity: Optimized arrays can detect as little as 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides

• Multiplexed analysis: The antibody can be used to probe protein chips containing multiple proteins to assess specificity and cross-reactivity

For comprehensive protein interaction studies, At3g51171 can be included in larger arrays with other Arabidopsis proteins. Research has demonstrated that properly validated antibodies show specific binding to their target proteins without cross-reacting with other proteins on the chip, including related protein family members .

A standardized protocol involves:

• Spotting recombinant proteins onto coated glass slides

• Blocking with appropriate buffer (typically 3% BSA)

• Probing with primary antibody at optimized dilution

• Detection with fluorescently-labeled secondary antibody

• Scanning using appropriate fluorescence detection systems

What methodological approaches can address epitope masking issues with At3g51171 antibody?

Epitope masking can significantly impact At3g51171 antibody performance, particularly in fixed tissues or certain experimental conditions. Advanced methodological approaches to resolve this include:

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER): Test buffers at pH ranges 6-9

  • Enzymatic epitope retrieval: Try proteinase K (1-20 μg/mL) or trypsin (0.1%)

  • Microwave vs. pressure cooker methods: Compare different heating devices

• Fixation optimization:

  • Test paraformaldehyde concentrations (1-4%) and incubation times

  • Compare cross-linking vs. precipitating fixatives

  • Evaluate post-fixation storage impact on epitope accessibility

• Detergent permeabilization:

  • Optimize detergent type (Triton X-100, NP-40, Tween-20) and concentration

  • Test permeabilization before or after fixation

  • Evaluate permeabilization time (10-30 minutes)

For protein complexes where At3g51171 may be involved in protein-protein interactions that mask epitopes, consider native vs. denaturing extraction conditions when preparing samples for analysis .

How can At3g51171 antibody be used in comparative cross-species studies?

While the At3g51171 antibody is specifically raised against Arabidopsis thaliana protein, its potential utility in cross-species applications requires careful methodological consideration:

• Sequence homology analysis: Before experimental testing, conduct bioinformatic analysis of protein sequence conservation between At3g51171 and homologs in target species

• Epitope mapping: If the immunizing peptide sequence is known, compare this region across species

• Graduated stringency testing: Begin with low-stringency conditions and increase washing stringency to optimize signal-to-noise ratio

• Validation approaches:

  • Western blot with recombinant proteins from each species

  • Peptide competition assays with species-specific peptides

  • Knockout/knockdown controls from each species when available

Cross-species reactivity should be experimentally validated rather than assumed. Even with high sequence homology, small differences in protein structure or post-translational modifications can significantly affect antibody binding .

What are the most common causes of false negative results with At3g51171 antibody, and how can they be addressed?

False negative results when using At3g51171 antibody can stem from multiple sources. Systematic troubleshooting approaches include:

Additionally, expression of At3g51171 may be tissue-specific or developmentally regulated. When investigating a new tissue or developmental stage, include positive control tissues with known At3g51171 expression . Performing RT-PCR in parallel can help confirm transcript presence when protein detection is negative.

How should researchers interpret unexpected molecular weight variations when detecting At3g51171?

Unexpected molecular weight variations when detecting At3g51171 require careful interpretation and can provide valuable biological insights:

• Post-translational modifications: Consider potential modifications like:

  • Phosphorylation (adds ~80 Da per site)

  • Glycosylation (can add 2-50+ kDa depending on modification)

  • Ubiquitination (adds ~8.5 kDa per ubiquitin)

  • Sumoylation (adds ~11 kDa per SUMO)

• Alternative splicing: Compare observed bands with predicted splice variants

  • Analyze genomic databases for annotated isoforms

  • Consider temporal or tissue-specific splicing patterns

• Proteolytic processing:

  • Compare N-terminal vs. C-terminal targeting antibodies

  • Use protease inhibitor cocktails during extraction

  • Consider physiological vs. extraction-induced processing

• Technical artifacts:

  • Incomplete denaturation causing altered migration

  • Protein aggregation or multimer formation

  • Interactions with other proteins surviving extraction

To systematically address this, map the epitope region of the antibody and compare with the region showing molecular weight variation. Additionally, perform parallel analysis with antibodies targeting different regions of At3g51171 when available .

What considerations are important when using At3g51171 antibody for co-immunoprecipitation of protein complexes?

When using At3g51171 antibody for co-immunoprecipitation (co-IP) of protein complexes, several critical methodological considerations must be addressed:

• Extraction buffer optimization:

  • Test different detergent types and concentrations

  • Optimize salt concentration to maintain complex integrity

  • Evaluate the impact of divalent cations (Ca²⁺, Mg²⁺)

  • Include appropriate phosphatase inhibitors to preserve interactions dependent on phosphorylation status

• Antibody coupling strategies:

  • Direct coupling to beads vs. capture with secondary antibody

  • Covalent vs. non-covalent coupling methods

  • Orientation-specific coupling to preserve antigen-binding regions

• Validation controls:

  • Perform reverse co-IP when possible

  • Include IgG isotype control

  • Compare results between crosslinked and non-crosslinked samples

  • Include negative control from tissues lacking At3g51171 expression

• Washing stringency optimization:

  • Test gradient of detergent concentrations

  • Establish salt concentration range maintaining specific interactions

  • Determine optimal number and duration of wash steps

The balance between preserving physiologically relevant protein interactions and reducing non-specific binding requires empirical testing for each complex of interest. Consider using mild crosslinking (0.1-1% formaldehyde) to stabilize transient interactions prior to extraction .

How can At3g51171 antibody be incorporated into advanced proteomics workflows?

Integration of At3g51171 antibody into advanced proteomics workflows enables deeper analysis of protein function, modification, and interaction networks:

• Antibody-based enrichment prior to mass spectrometry:

  • Immunoprecipitation followed by LC-MS/MS analysis

  • Sequential IP to isolate specific sub-complexes

  • Phospho-enrichment to study At3g51171 phosphorylation states

• Proximity labeling approaches:

  • Antibody-directed enzyme proximity labeling (APEX or BioID fusion constructs)

  • Identification of transient or weak interactors in native cellular contexts

  • Analysis of spatial proteomics surrounding At3g51171

• Single-cell proteomics integration:

  • Antibody-based sorting of specific cell populations

  • Validation of mass spectrometry findings at single-cell resolution

  • Correlation of protein expression with phenotypic variations

• Quantitative multiplex approaches:

  • Multiplexed immunofluorescence using At3g51171 antibody with other markers

  • Sequential elution of epitopes for iterative detection of multiple proteins

  • Integration with spatial transcriptomics data

These advanced applications typically require thorough validation of antibody specificity, ideally using protein microarray approaches that can test cross-reactivity against numerous potential Arabidopsis proteins simultaneously .

What considerations are important when developing nanobody alternatives to conventional At3g51171 antibodies?

Development of nanobody alternatives to conventional At3g51171 antibodies represents an emerging research direction with several important methodological considerations:

• Nanobody generation and selection approach:

  • Immunization of camelids (alpacas, llamas) with purified At3g51171

  • Phage display selection using varying stringency conditions

  • Yeast display for quantitative affinity maturation

  • Rational design based on protein structure prediction

• Epitope selection strategy:

  • Target conserved vs. variable regions depending on application needs

  • Consider epitope accessibility in native protein conformation

  • Evaluate potential for blocking protein-protein interactions

• Engineering considerations:

  • Fusion tag strategies (Fc, albumin) to extend half-life or alter function

  • CDR optimization for improving specificity and affinity

  • Humanization strategies for potential therapeutic applications

• Validation approaches:

  • Comparative analysis with conventional antibodies

  • Cross-reactivity profiling using protein arrays

  • Functional blocking studies in cellular contexts

How can At3g51171 antibody be used in conjunction with CRISPR-based genome editing to study protein function?

Integrating At3g51171 antibody with CRISPR-based genome editing creates powerful tools for functional protein characterization:

• Validation of gene editing outcomes:

  • Protein-level confirmation of knockout efficiency

  • Detection of truncated proteins from partial knockouts

  • Identification of compensatory protein expression changes

• Tagged endogenous protein studies:

  • Verification of successful epitope tag integration

  • Comparison of tagged vs. untagged protein expression levels

  • Analysis of tag impact on protein localization and function

• Temporal protein dynamics analysis:

  • Combination with inducible CRISPR systems for time-course studies

  • Protein degradation rate measurement after inducible knockout

  • Comparison of transcript vs. protein persistence after gene targeting

• Protein variant characterization:

  • Analysis of protein expression from CRISPR-introduced point mutations

  • Comparison of protein stability between wild-type and variant forms

  • Assessment of modification patterns in protein variants

• Multi-modal phenotypic analysis:

  • Correlation of protein levels with phenotypic outcomes

  • Single-cell analysis of protein variation in edited populations

  • Integration with transcriptomic and metabolomic data from edited lines

When combining antibody detection with CRISPR studies, it's crucial to confirm antibody epitope preservation in edited proteins. For comprehensive studies, combining antibodies targeting different protein regions provides validation and deeper functional insights .

What methodological approaches can improve reproducibility when using At3g51171 antibody across different experimental systems?

Ensuring reproducibility with At3g51171 antibody across different experimental systems requires systematic standardization of multiple parameters:

• Antibody validation and characterization:

  • Lot-to-lot testing with standard samples

  • Quantitative assessment of binding affinity

  • Epitope mapping to understand targeting specificity

  • Cross-reactivity profiling against related proteins

• Sample preparation standardization:

  • Consistent extraction buffer composition

  • Standardized tissue collection and processing times

  • Controlled protein quantification methods

  • Validated protein denaturation protocols

• Assay optimization documentation:

  • Detailed recording of all optimization parameters

  • Development of positive control standards

  • Creation of calibration curves for quantitative applications

  • Inter-laboratory validation studies

• Data analysis pipelines:

  • Standardized image acquisition settings

  • Consistent quantification methods

  • Appropriate statistical analyses for biological variability

  • Transparent data normalization approaches

Research has shown that antibody performance can vary significantly between applications and experimental conditions. Thorough validation using approaches like protein microarrays can confirm specificity against multiple potential cross-reactive proteins simultaneously . For critical applications, parallel validation with orthogonal methods like mass spectrometry provides additional confidence in results.

How should researchers approach experimental design when At3g51171 antibody shows weak or variable detection?

When At3g51171 antibody yields weak or variable detection, a systematic experimental redesign approach is necessary:

• Comprehensive antibody performance evaluation:

  • Test multiple concentration ranges (0.1-20 μg/mL)

  • Evaluate different incubation conditions (1 hr at RT vs. overnight at 4°C)

  • Compare detection methods (chemiluminescence, fluorescence, colorimetric)

  • Assess sensitivity using purified recombinant protein standard curve

• Sample preparation optimization:

  • Test multiple extraction methods (native vs. denaturing)

  • Evaluate protein enrichment approaches (subcellular fractionation, immunoprecipitation)

  • Optimize protein loading amounts (10-100 μg total protein)

  • Assess impact of protease inhibitor cocktails

• Biological considerations:

  • Verify expression levels by transcript analysis

  • Determine developmental or stress-responsive expression patterns

  • Evaluate protein stability and turnover rates

  • Consider post-translational modifications affecting epitope recognition

• Technical alternatives:

  • Test alternative antibodies targeting different epitopes

  • Consider developing epitope-tagged constructs

  • Evaluate mass spectrometry-based detection methods

  • Explore proximity labeling approaches

For optimal detection, polyclonal antibodies targeting At3g51171 typically work best at concentrations between 1.7-15 μg/mL, which is generally lower than concentrations needed for monoclonal antibodies (5-25 μg/mL) . Signal amplification systems like tyramide signal amplification can enhance detection of low-abundance proteins while maintaining specificity.

How might protein engineering approaches expand the functionality of At3g51171 antibodies?

Protein engineering offers several promising approaches to enhance At3g51171 antibody functionality:

• Fragment-based engineering:

  • Generation of Fab or F(ab')₂ fragments for improved tissue penetration

  • Development of bispecific antibodies targeting At3g51171 and interacting proteins

  • Creation of antibody-enzyme fusion proteins for proximity labeling applications

• Affinity and specificity optimization:

  • CDR mutagenesis to enhance binding properties

  • Directed evolution for improved specificity

  • Computational design of binding interfaces

• Functional modification:

  • Integration of photocrosslinking amino acids for covalent target capture

  • Development of conditional binding antibodies (pH, redox, or light-responsive)

  • Creation of allosteric sensor antibodies for conformational studies

• Detection enhancement:

  • Site-specific conjugation of fluorophores or other detection tags

  • Quantum dot or nanoparticle conjugation for enhanced sensitivity

  • Design of split-antibody complementation systems for proximity studies

These approaches draw on recent advances in antibody engineering, including new methods to develop nanobody antagonists with tunable pharmacological properties . In particular, the development of heavy chain-only antibody fragments (nanobodies) offers advantages for accessing epitopes unavailable to conventional antibodies and enhancing tissue penetration.

What emerging methodologies might enhance At3g51171 antibody applications in plant cell biology?

Emerging methodologies poised to transform At3g51171 antibody applications in plant cell biology include:

• Advanced microscopy integration:

  • Super-resolution microscopy techniques (STORM, PALM, STED)

  • Expansion microscopy for enhanced spatial resolution

  • Correlative light and electron microscopy (CLEM)

  • Light-sheet microscopy for dynamic 3D imaging

• Single-cell applications:

  • Microfluidic-based single-cell Western blotting

  • Mass cytometry (CyTOF) for multiparameter protein analysis

  • Spatial transcriptomics integration with protein localization

  • Droplet-based single-cell proteomics

• In vivo tracking methodologies:

  • Optogenetic protein targeting and manipulation

  • Development of plant-optimized intrabodies

  • Targeted protein degradation approaches (PROTAC-like)

  • Genetically encoded sensors coupled with antibody detection

• Synthetic biology interfaces:

  • Antibody-based biosensors for metabolite detection

  • Engineered protein circuits with antibody-based readouts

  • Cell-free expression systems for rapid protein characterization

  • Nanobody-based orthogonal signaling systems