At1g78860 Antibody

What is At1g78860 and what is its function in Arabidopsis thaliana?

At1g78860 encodes a mannose-binding lectin family protein in Arabidopsis thaliana, also known as EP1-like glycoprotein 4, F9K20.9, or curculin-like protein. This protein contains a GNA-related lectin domain that localizes to the plasma membrane . Functionally, it plays several important roles:

• Defense responses: It is involved in plant cell death and defense regulation through induction of downstream defense-related genes and salicylic acid pathways .

• Glycan binding: Glycan array screening shows that the protein has affinity toward Manα and/or Manβ and GalNAc residues .

• Boron stress responses: A T-DNA insertion mutant in the exon of At1g78860 (SALK_144144C) exhibited significantly higher tolerance to low-boron (0.1 μM) stress compared to wild-type Col-0, suggesting a role in boron homeostasis .

• Guard cell signaling: Expression profiling shows At1g78860 is differentially expressed in guard cells, suggesting potential involvement in stomatal functions .

When sourcing antibodies against At1g78860, researchers should consider:

• Antibody type: Both polyclonal and monoclonal antibodies are available. Polyclonal antibodies provide higher sensitivity but may have batch-to-batch variation, while monoclonal antibodies offer higher specificity .

• Target epitope: The GNA-related lectin domain is essential for the protein's function and binding to D-mannose, making it a critical region for antibody recognition .

• Validation data: Request comprehensive validation data including Western blot results against wild-type and knockout plant tissues .

• Cross-reactivity: Verify the antibody doesn't cross-react with other lectins or related proteins in your experimental system .

• Application compatibility: Ensure the antibody is validated for your specific application (Western blot, ELISA, immunoprecipitation, etc.) .

How can At1g78860 antibodies be used to study plant-pathogen interactions?

At1g78860 antibodies have proven valuable in dissecting plant immunity mechanisms:

• Pathogen response monitoring: The antibody can be used to track At1g78860 protein levels during infection with pathogens such as Xanthomonas campestris pv. vesicatoria (Xcv) in pepper plants or Pseudomonas syringae pv. tomato DC3000 in Arabidopsis .

• Interactome analysis: Co-immunoprecipitation coupled with mass spectrometry has revealed At1g78860 protein interaction networks. When conducting such experiments:

  • Use freshly prepared plant tissues

  • Include appropriate negative controls (e.g., GFP-only immunoprecipitation)

  • Perform on-bead or in-gel trypsin digestion for mass spectrometry analysis

• Subcellular localization: Immunohistochemistry with At1g78860 antibodies can reveal dynamic changes in protein localization during pathogen challenge .

• Signaling pathway analysis: Western blotting with phospho-specific antibodies can help determine the activation status of At1g78860 during immune responses .

A robust experimental design should include appropriate time points post-infection (typically 0, 6, 12, 24, 48 hours) and parallel qRT-PCR analysis to correlate protein and transcript levels.

What techniques ensure proper validation of At1g78860 antibody specificity?

Comprehensive validation requires multiple approaches:

• Western blot analysis with positive and negative controls:

  • Wild-type plants (positive control)

  • At1g78860 T-DNA insertion mutants (SALK_144144C) (negative control)

  • Recombinant At1g78860 protein (positive control)

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish signal in Western blots or immunostaining .

• Cross-validation with orthogonal methods:

  • Compare protein detection with transcript abundance by qRT-PCR

  • Use GFP-tagged At1g78860 expressed in plants and detect with both anti-GFP and anti-At1g78860 antibodies

• Testing across experimental conditions:

  • Verify specificity in multiple tissue types and under various treatments

  • Test detection in plant extracts with different protein extraction methods

• Mass spectrometry confirmation: Immunoprecipitate At1g78860 using the antibody and confirm identity by mass spectrometry analysis .

How does post-translational modification affect At1g78860 antibody recognition?

At1g78860 is a glycoprotein with a GNA-related lectin domain, and its post-translational modifications (PTMs) can significantly impact antibody recognition:

• Glycosylation effects: The mannose-binding lectin domains may undergo glycosylation that can mask epitopes. Studies have shown that:

  • Differential glycosylation patterns exist between stressed and non-stressed plants

  • Deglycosylation treatments prior to Western blotting may be necessary for consistent detection

• Phosphorylation status: During plant defense responses, phosphorylation events may alter protein conformation and antibody accessibility .

• Methodological approaches:

  • Use phosphatase treatment to assess phosphorylation effects

  • Compare native versus denatured protein detection efficiency

  • Consider developing modification-specific antibodies for studying particular PTM states

• Conformational considerations: The GNA-related lectin domain conformation is essential for binding to D-mannose, and antibodies targeting this region may show differential recognition depending on ligand binding status .

What are the optimal experimental conditions for immunoprecipitation with At1g78860 antibodies?

Successful immunoprecipitation of At1g78860 requires optimization of several parameters:

• Buffer composition:

  • Use 50% glycerol, 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as preservative

  • Include protease inhibitor cocktails to prevent degradation

  • Consider adding phosphatase inhibitors if studying phosphorylation-dependent interactions

• Sample preparation:

  • For plant tissues, grind in liquid nitrogen before adding extraction buffer

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

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Antibody binding conditions:

  • Incubate antibody with sample overnight at 4°C with gentle rotation

  • Use 2-5 μg antibody per 100-500 μg total protein

  • Include negative controls (non-immune IgG or extracts from knockout plants)

• Washing conditions:

  • Perform 3-5 washes with increasingly stringent buffers

  • Monitor washing efficiency by measuring protein concentration in wash fractions

• Elution and analysis:

  • Elute with either acidic conditions (glycine, pH 2.5) or SDS sample buffer

  • For interaction studies, consider on-bead trypsin digestion for mass spectrometry analysis

Research has shown that co-immunoprecipitation followed by mass spectrometry can identify At1g78860 interaction partners involved in plant immunity pathways .

How can At1g78860 antibodies be used to investigate boron deficiency tolerance mechanisms?

At1g78860 has been identified as a potential regulator of boron deficiency tolerance in Arabidopsis:

• Experimental approaches:

  • Compare At1g78860 protein levels between wild-type and the low-boron tolerant (lbt) mutant (SALK_144144C) using Western blotting

  • Track protein expression during boron deficiency time courses

  • Perform co-immunoprecipitation to identify interaction partners under normal versus boron-deficient conditions

• Tissue-specific analysis:

  • Examine At1g78860 protein localization in roots versus shoots under boron stress

  • Compare protein expression patterns in boron-efficient versus boron-inefficient Arabidopsis accessions

• Integration with transcriptomic data:

  • Correlate protein levels with the significant upregulation (3.4-fold) of At1g78860 observed in guard cells under stress conditions

  • Investigate protein-level consequences of the SNPs associated with At1g78860 in natural Arabidopsis populations

What methodological considerations are important when using At1g78860 antibodies in different plant species?

When extending At1g78860 antibody applications to non-Arabidopsis species:

• Cross-reactivity assessment:

  • Perform Western blot analysis using tissues from the target species

  • Include positive (Arabidopsis) and negative (knockout) controls

  • Consider sequence homology between species when interpreting results

• Epitope conservation analysis:

  • Align protein sequences from different species to identify conserved regions

  • Select antibodies targeting highly conserved epitopes for cross-species applications

  • For pepper plants, which have the homologous CaMBL1 protein, validate specificity in Capsicum annuum tissues

• Optimization of extraction protocols:

  • Adjust buffer compositions to account for species-specific differences in secondary metabolites

  • Modify tissue disruption methods based on plant structure

  • Consider species-specific protease inhibitor requirements

• Citrus applications:

  • When studying citrus rootstock responses to pathogens like Candidatus Liberibacter asiaticus, additional validation steps are required due to high levels of inhibitory compounds

How can At1g78860 antibodies contribute to understanding G-protein coupled signaling in plants?

At1g78860 has been implicated in plant signaling networks regulated by G-proteins:

• Experimental approaches:

  • Use At1g78860 antibodies to compare protein levels in wild-type versus gpa1-5 and gcr1-5 mutants

  • Perform co-immunoprecipitation to detect potential interactions with G-protein subunits

  • Conduct immunolocalization studies to determine if At1g78860 co-localizes with G-protein signaling components

• Signaling pathway analysis:

  • The study by GCR1 and GPA1 coupling showed that At1g78860 was significantly up-regulated in the double mutant, suggesting a role in G-protein regulated defense pathways

  • Use antibodies to verify if this transcriptional change corresponds to protein-level alterations

• Integration with hormone signaling:

  • Investigate whether At1g78860 protein levels change in response to hormone treatments

  • Analyze protein expression in hormone signaling mutants to position At1g78860 within signaling cascades

What are common challenges when using At1g78860 antibodies and how can they be addressed?

Researchers frequently encounter several issues when working with plant protein antibodies:

• High background in Western blots:

  • Solution: Optimize blocking conditions (try 2.5% skimmed milk or 3-5% BSA)

  • Solution: Increase washing stringency and duration

  • Solution: Dilute primary antibody (1:1000-1:5000) and secondary antibody (1:5000-1:10000)

• Weak or absent signal:

  • Solution: Enrich target protein through subcellular fractionation

  • Solution: Use enhanced chemiluminescence (ECL) detection systems

  • Solution: Ensure plant material is fresh and processed quickly to minimize degradation

• Multiple bands or unexpected band sizes:

  • Solution: Include a peptide competition assay to identify specific bands

  • Solution: Use protein extracts from knockout plants as negative controls

  • Solution: Consider the presence of post-translational modifications or protein isoforms

• Poor immunoprecipitation efficiency:

  • Solution: Cross-link antibody to beads to prevent antibody co-elution

  • Solution: Optimize binding conditions (temperature, time, buffer composition)

  • Solution: Pre-clear lysates to reduce non-specific binding

• Inconsistent results between experiments:

  • Solution: Standardize protein extraction methods

  • Solution: Use internal loading controls consistently

  • Solution: Aliquot antibodies to avoid freeze-thaw cycles

How should researchers interpret contradictory results between transcript and protein levels of At1g78860?

Discrepancies between mRNA and protein levels are common in plant research and require careful interpretation:

• Methodological verification:

  • Confirm primer specificity for qRT-PCR

  • Validate antibody specificity as described in section 2.2

  • Ensure appropriate normalization for both techniques

• Biological explanations to consider:

  • Post-transcriptional regulation: At1g78860 may be subject to miRNA-mediated regulation

  • Protein stability differences: Environmental conditions may affect protein turnover without changing transcript levels

  • Temporal dynamics: Transcript and protein peaks may occur at different time points

• Experimental approach for reconciliation:

  • Perform time-course experiments measuring both transcript and protein levels

  • Use translation inhibitors to assess protein half-life

  • Consider polysome profiling to assess translation efficiency

• Case study example: In research examining SARS-CoV-2 antibodies, there was a notable discrepancy between binding affinity (ELISA) and neutralizing capacity of antibodies, which was attributed to differences in protein structure between plate-bound and native conformations . Similar phenomena may explain At1g78860 discrepancies.

How might emerging antibody technologies enhance At1g78860 research?

Advances in antibody technology offer new opportunities for At1g78860 research:

• AI-designed antibodies:

  • Machine learning approaches have successfully designed antibodies with improved cross-binding to different antigen variants

  • Similar approaches could be applied to develop At1g78860 antibodies with enhanced specificity or broader cross-species reactivity

• Single-domain antibodies (nanobodies):

  • Their small size allows better penetration into plant tissues

  • May provide access to epitopes inaccessible to conventional antibodies

  • Can be expressed in planta for real-time protein tracking

• Multiplexed detection systems:

  • Antibody arrays could simultaneously monitor At1g78860 and interacting partners

  • Proximity ligation assays could visualize protein-protein interactions in situ

• Modification-specific antibodies:

  • Development of antibodies specific to phosphorylated or glycosylated forms of At1g78860

  • Would enable tracking of post-translational modification dynamics during stress responses

What novel experimental applications could be developed for At1g78860 antibodies?

Innovative applications that extend beyond traditional uses include:

• CRISPR-based tagging combined with antibody detection:

  • Use CRISPR to tag endogenous At1g78860 with epitope tags

  • Employ both tag-specific and At1g78860-specific antibodies for validation and functional studies

• Single-cell protein analysis:

  • Apply At1g78860 antibodies in emerging single-cell proteomics workflows

  • Could reveal cell-type specific expression patterns in complex tissues

• Biosensor development:

  • Engineer antibody-based biosensors for real-time monitoring of At1g78860 in live plants

  • Could provide dynamic information about protein responses to various stresses

• Synthetic biology applications:

  • Use antibodies to monitor engineered At1g78860 variants with enhanced functions

  • Support development of plants with improved stress tolerance or pathogen resistance

• Structural biology integration:

  • Employ antibodies as crystallization chaperones for structural studies of At1g78860

  • Would advance understanding of mannose-binding mechanisms and ligand interactions