At1g66620 Antibody

What is At1g66620 and why is it significant in plant research?

At1g66620 is a gene encoding a RING-finger domain protein in Arabidopsis thaliana. This protein belongs to a significantly overrepresented class of proteins in Arabidopsis compared to other eukaryotic genomes (1.42% of Arabidopsis proteins contain RING domains versus 0.7-0.75% in Drosophila, C. elegans, and yeast) . The RING-finger domain functions primarily as a protein-interaction domain, and RING-finger proteins are implicated in diverse biological processes including transcriptional regulation, translational regulation, and targeted proteolysis . Many RING proteins demonstrate biochemical ubiquitin ligase activity, suggesting potential roles in protein degradation pathways .

Research on At1g66620 is particularly valuable because it belongs to a protein family with potential functional redundancy, making it an interesting target for understanding compensatory mechanisms in plant biology . Developing antibodies against this protein enables researchers to investigate its expression patterns, protein interactions, and functional roles in plant development and stress responses.

What types of antibodies are most suitable for At1g66620 detection in plant tissues?

For researching At1g66620 in plant tissues, researchers typically need to choose between several antibody formats:

• Monoclonal antibodies: Provide high specificity but may have limited detection sensitivity (approximately 1.0 ppb in similar systems)

• Recombinant antibody fragments: Offer superior sensitivity, with monomeric fragments achieving detection limits down to 0.1 ppb (10× more sensitive than conventional monoclonals)

• Single-chain antibody fragments (scAb): These fusion proteins containing single-chain Fv with a CK domain have demonstrated excellent performance in plant protein detection systems

When studying RING-finger proteins like At1g66620, recombinant antibody fragments often provide the best combination of specificity and sensitivity. The Escherichia coli expression system allows for production of both monomeric and dimeric antibody species that can be purified through nickel chelate immunosorbent columns or by immunoaffinity purification using a constant domain tag .

How do I verify At1g66620 antibody specificity in Arabidopsis samples?

Verifying antibody specificity for At1g66620 requires multiple control experiments:

Primary specificity controls:

• Western blot analysis comparing wild-type plants with At1g66620 knockout/knockdown lines

• Immunoprecipitation followed by mass spectrometry identification

• Pre-absorption tests with recombinant At1g66620 protein

• Cross-reactivity assessment with closely related RING-finger proteins

Secondary validation methods:

• Protein localization consistency with predicted subcellular targeting

• Expression patterns matching transcript data from RNA-seq or microarray studies

• Comparison of results using multiple antibodies targeting different epitopes of At1g66620

How should immunoassays be optimized for detecting At1g66620 in complex plant extracts?

When developing immunoassays for At1g66620 detection in complex plant extracts, researchers should consider both direct and indirect assay formats:

Table 1: Comparison of Immunoassay Formats for At1g66620 Detection

Based on comparative studies with similar protein targets, monomeric antibody fragments consistently provide greater sensitivity than either dimeric fragments or intact monoclonal antibodies in both assay formats . When optimizing the assay, consider:

• Buffer composition to minimize plant extract interference

• Blocking agents that reduce non-specific binding without affecting antigen recognition

• Incubation temperatures and times optimal for antibody-antigen interactions

• Detection methods appropriate for expected protein abundance levels

For low-abundance RING-finger proteins like At1g66620, enhanced chemiluminescence or amplified colorimetric detection systems may be necessary to achieve adequate sensitivity while maintaining specificity.

What are the advanced approaches for studying At1g66620 protein interactions using antibody-based methods?

RING-finger proteins function primarily through protein-protein interactions . Advanced antibody-based methods to study At1g66620 interactions include:

• Co-immunoprecipitation (Co-IP) with targeted optimizations:

  • Use crosslinking agents to capture transient interactions

  • Implement two-step IP protocols to increase specificity

  • Apply quantitative mass spectrometry for interaction partner identification

• Proximity-based labeling combined with immunoprecipitation:

  • Fuse At1g66620 with BioID or APEX2 for proximity labeling

  • Use At1g66620 antibodies to verify expression and localization

  • Combine with ubiquitination assays to identify potential substrates

• Antibody-based protein complementation assays:

  • Split reporter systems with antibody validation

  • FRET/BRET approaches using antibody-verified constructs

  • Multicolor co-localization with antibody staining

When designing these experiments, it's critical to account for the potential redundancy among RING-domain proteins in Arabidopsis . Multiple controls including inactive RING-domain mutants should be incorporated to distinguish specific from non-specific interactions.

How can bispecific antibodies enhance At1g66620 research applications?

Bispecific antibodies (bsAbs) targeting At1g66620 alongside another protein of interest offer innovative research applications:

• Simultaneous detection of At1g66620 and potential interaction partners:
  Bispecific antibodies can be engineered to bind both At1g66620 and suspected interaction partners, facilitating co-localization studies without requiring protein tagging or overexpression .

• Enhanced immunoprecipitation for low-abundance complexes:
  By targeting At1g66620 and a known complex component simultaneously, bsAbs can increase the efficiency of pulling down low-abundance protein complexes .

• Monitoring dynamic protein-protein interactions:
  Strategically designed bsAbs can enable detection of conformational changes or interaction-dependent epitope accessibility .

When developing bispecific antibodies for At1g66620 research, consider the molecular geometry carefully, as this significantly impacts functionality . As demonstrated in recent studies, bsAbs with the same building blocks but different molecular geometry can exhibit varying activity levels . Integration of single-domain antibodies (sdAbs) onto IgG scaffolds offers versatility but requires optimization of the fusion site and linker design to maintain stability and binding efficiency .

What expression systems yield the highest quality antibodies against plant RING-finger proteins like At1g66620?

Based on comparative analysis, the following expression systems have demonstrated success for antibodies targeting plant RING-finger proteins:

Table 2: Expression Systems for At1g66620 Antibody Production

For antibody fragments targeting At1g66620, E. coli systems have shown particular success, allowing expression of both monomeric and dimeric antibody species that can be purified through nickel chelate columns or immunoaffinity purification . The monomeric fragments consistently demonstrate superior sensitivity compared to conventional formats, reaching detection limits as low as 0.1 ppb .

What epitope selection strategies maximize specificity for At1g66620 while minimizing cross-reactivity with other RING-domain proteins?

Epitope selection is particularly challenging for At1g66620 due to the abundance of RING-domain proteins in Arabidopsis and their potential functional redundancy . Optimal strategies include:

• Computational analysis phase:

  • Perform multiple sequence alignment of all Arabidopsis RING-finger proteins

  • Identify unique regions outside the conserved RING domain

  • Use structural prediction to identify surface-exposed, unique peptide regions

  • Evaluate uniqueness through BLAST searches against the Arabidopsis proteome

• Experimental validation phase:

  • Test multiple candidate epitopes through peptide immunization approaches

  • Evaluate epitope accessibility in native protein through structural techniques

  • Screen antibodies against knockout lines and recombinant protein arrays

• Advanced specificity enhancement:

  • Implement negative selection strategies during antibody development

  • Use epitope masking with related RING proteins during screening

  • Consider dual-epitope recognition strategies to increase specificity

For At1g66620, regions outside the RING domain offer better specificity targets, as the RING domain itself contains eight conserved cysteine and histidine residues in a cross-brace arrangement that is highly similar across family members .

How do different antibody formats compare in their ability to detect native At1g66620 in plant cells?

Different antibody formats exhibit varying capabilities for detecting native At1g66620 in plant samples:

Table 3: Comparative Performance of Antibody Formats for At1g66620 Detection

Research comparing recombinant antibody fragments to conventional formats demonstrates that monomeric fragments generally provide the highest sensitivity in both direct and indirect assay formats . Their smaller size may also offer advantages for tissue penetration in immunohistochemistry applications, although this benefit must be balanced against potentially reduced stability in some plant extract conditions.

For challenging applications like chromatin immunoprecipitation or in situ protein complex detection, specialized approaches may be necessary. Some researchers have found success with chemical crosslinking followed by epitope retrieval to improve accessibility of At1g66620 in its native protein complexes.

How should researchers address inconsistent At1g66620 antibody performance across different plant tissues?

Inconsistent antibody performance when detecting At1g66620 across different plant tissues may result from several factors:

• Tissue-specific protein modifications:

  • RING-finger proteins like At1g66620 may undergo tissue-specific post-translational modifications

  • Ubiquitination, phosphorylation, or other modifications may mask epitopes

  • Solution: Try multiple antibodies targeting different epitopes; use denaturing conditions when appropriate

• Variable protein complex formation:

  • At1g66620 likely functions in protein complexes that differ between tissues

  • Epitope accessibility may be affected by interaction partners

  • Solution: Include detergent screening in your protocol development; test gentle dissociation methods

• Extraction efficiency variations:

  • Different tissues contain varying levels of interfering compounds

  • RING proteins may have different subcellular distributions across tissues

  • Solution: Optimize extraction buffers for each tissue type; consider subcellular fractionation

• Protocol adaptation guidance:
  When adapting protocols between tissues, systematically modify:

  • Fixation times and temperatures

  • Antigen retrieval methods

  • Blocking reagents (consider tissue-specific autofluorescence or peroxidase activity)

  • Detection system sensitivity

RING-finger domain proteins show varying expression patterns across tissues and developmental stages , so validation of antibody performance should include tissue-specific controls and careful titration of antibody concentrations.

What are the most common pitfalls in analyzing At1g66620 expression data from antibody-based experiments?

When analyzing At1g66620 expression data, researchers should be aware of these common pitfalls:

• Misinterpreting signal due to cross-reactivity:

  • The Arabidopsis genome encodes 387 domains with potential to form RING-type cross-brace structures

  • Many RING domains overlap with predicted PHD domains, complicating specificity

  • Solution: Always validate with genetic controls (knockouts/knockdowns); confirm with orthogonal methods

• Overlooking functional redundancy effects:

  • The significant abundance of RING-domain proteins in Arabidopsis suggests functional redundancy

  • Manipulating At1g66620 levels may trigger compensatory changes in related proteins

  • Solution: Monitor multiple family members simultaneously; consider combinatorial genetic approaches

• Misattributing cellular localization:

  • At1g66620 may shuttle between cellular compartments or exist in multiple pools

  • Fixation methods can alter apparent distribution

  • Solution: Compare multiple fixation methods; use live-cell imaging with validated tags when possible

• Data normalization challenges:

  • Common loading controls may not remain constant under conditions affecting At1g66620

  • Tissue-specific expression complicates comparison between samples

  • Solution: Use multiple loading controls; consider absolute quantification with recombinant standards

When publishing At1g66620 research, provide detailed methodology including antibody validation data, extraction conditions, and imaging parameters to ensure reproducibility.

How can contradictory results from different At1g66620 antibody-based methods be reconciled?

When faced with contradictory results from different antibody-based methods targeting At1g66620:

• Systematic comparison of epitopes and antibody properties:

  • Map the epitopes recognized by each antibody

  • Evaluate whether epitopes might be differentially accessible in various experimental contexts

  • Compare antibody formats (monomeric vs. dimeric fragments vs. full IgG)

• Method-specific considerations:

  • Western blot: Denaturation may expose epitopes hidden in native conditions

  • Immunoprecipitation: Buffer conditions affect complex stability

  • Immunohistochemistry: Fixation chemistry can modify epitopes

• Resolution strategies:

  • Develop correlation matrices between methods and antibodies

  • Implement orthogonal non-antibody techniques (mass spectrometry, RNA analysis)

  • Generate epitope-tagged versions of At1g66620 for method validation

  • Use CRISPR-based tagging of endogenous At1g66620 as a reference standard

• Biophysical characterization:

  • Assess antibody binding parameters (affinity, on/off rates) under each method's conditions

  • Consider whether post-translational modifications affect each antibody differently

  • Evaluate temperature and pH sensitivity of each antibody-epitope interaction

Given that different antibody formats demonstrate varying sensitivity levels (monomeric fragments showing ~10× greater sensitivity than conventional antibodies) , quantitative differences between methods may reflect these inherent sensitivity variations rather than true biological differences.

How can cutting-edge antibody engineering approaches enhance At1g66620 research beyond conventional applications?

Advanced antibody engineering offers new possibilities for At1g66620 research:

• Proximity-dependent labeling with antibody-enzyme fusions:

  • Fusing peroxidase or biotin ligase enzymes to At1g66620-specific antibody fragments

  • Enabling identification of transient interaction partners in native contexts

  • Preserving endogenous expression levels unlike traditional fusion protein approaches

• Intrabodies for tracking dynamic At1g66620 behaviors:

  • Developing antibody fragments optimized for intracellular expression

  • Allowing visualization of At1g66620 dynamics without overexpression artifacts

  • Potentially interfering with specific interactions while preserving others

• Nanobody-based degradation systems:

  • Creating plant-optimized nanobody-based degradation systems targeting At1g66620

  • Enabling rapid, conditional depletion of endogenous protein

  • Providing temporal control not possible with genetic knockouts

• Bispecific antibody applications:

  • Developing bispecific formats to study At1g66620 in complex with specific partners

  • Creating forced proximity through antibody-mediated bridging

  • Testing functional redundancy through simultaneous targeting of multiple family members

When designing advanced antibody applications, molecular geometry becomes particularly important . Studies have shown that constructs with identical binding domains but different geometries exhibit varying activities . For At1g66620 research, optimizing both internal constraints (steric hindrance between binding domains) and external constraints (target accessibility in complexes) is essential for successful application .

What experimental design considerations are critical when using At1g66620 antibodies to study ubiquitin ligase activity?

When designing experiments to study At1g66620's potential ubiquitin ligase activity:

• Activity preservation strategies:

  • RING-finger proteins often function as E3 ubiquitin ligases

  • Antibody binding may interfere with this enzymatic activity

  • Critical considerations include epitope location relative to the catalytic domain and binding conditions that preserve zinc coordination

• Experimental setup for ubiquitination assays:

  • Use non-interfering antibodies for pull-down/immunoprecipitation

  • Implement controls with RING-domain mutants lacking ligase activity

  • Include both substrate-independent (autoubiquitination) and substrate-dependent assays

• Temporal dynamics consideration:

  • Ubiquitination is often transient and condition-dependent

  • Design time-course experiments with appropriate inhibitors

  • Consider cell-free systems where reaction components can be controlled

• Technical requirements:

  • Preserving native protein complexes during extraction

  • Preventing deubiquitinase activity during sample processing

  • Distinguishing different ubiquitin chain topologies in analysis

Because RING-finger domains coordinate zinc ions through cysteine and histidine residues in a cross-brace structure , buffer conditions must be carefully optimized to maintain domain integrity while allowing effective antibody binding.

What emerging technologies are most promising for expanding At1g66620 antibody applications in plant science?

Several emerging technologies show particular promise for At1g66620 antibody applications:

• Microfluidic antibody-based single-cell analysis:

  • Integration of plant protoplast isolation with microfluidic antibody-based detection

  • Enabling cell-type-specific analysis of At1g66620 expression and modification

  • Potential for correlating protein levels with transcriptomics at single-cell resolution

• Advanced imaging with engineered antibody fragments:

  • Super-resolution microscopy compatible antibody fragments

  • Expansion microscopy protocols optimized for plant tissues

  • Multi-parameter imaging with orthogonal antibody labeling systems

• Antibody-enabled spatial proteomics:

  • Combining antibody-based enrichment with spatially-resolved mass spectrometry

  • Mapping At1g66620 interaction networks across tissue domains

  • Correlating with transcriptomic and metabolomic spatial data

• Synthetic biology applications:

  • Antibody-based biosensors for monitoring At1g66620 activity in real-time

  • Engineered circuits using antibody-based modulation of At1g66620 function

  • Plant-optimized nanobodies as selective inhibitors or activators

• Artificial intelligence for antibody design:

  • AI-guided epitope selection for increased specificity against At1g66620

  • Structure-based optimization of antibody binding properties

  • Prediction of cross-reactivity across the RING protein family

With the high abundance of RING-domain proteins in Arabidopsis (1.42% of the proteome) , computational approaches to enhance antibody specificity will be particularly valuable for advancing At1g66620 research.