At5g39180 Antibody

What is At5g39180 and why is it significant for plant defense research?

At5g39180 is a germin-like protein in Arabidopsis thaliana that functions as a secreted Mn-binding protein with an important role in plant defense mechanisms. This protein belongs to the RmlC-like cupins superfamily and has been identified as differentially regulated in various stress and defense-related experimental conditions .

According to transcriptomic analyses, At5g39180 is among the genes down-regulated in BAK1 (Y610F)-Flag plants compared to wild-type BAK1, with a fold change of 0.88 (log2) or 1.84 (FC) as shown in the following data:

*The asterisk identifies this gene as selected for qPCR analysis in studies examining BAK1 function in plant immunity .

Methodologically, researchers interested in At5g39180 often study its expression patterns in response to immune elicitors like flg22 or elf18, as well as its potential role in PAMP-triggered immunity (PTI) signaling cascades.

How should I design experiments to validate At5g39180 antibody specificity?

Validating antibody specificity for At5g39180 requires a multi-step approach following established antibody validation protocols:

• Expected localization testing: Confirm that the antibody produces staining patterns consistent with the expected subcellular localization of At5g39180 (secreted protein).

• Quantitative titration: Determine optimal antibody concentration through serial dilutions, typically starting at 1:1000 for Western blots based on similar germin-like protein antibodies .

• Orthogonal validation: Compare protein detection using alternative methods such as:

  • Mass spectrometry validation of immunoprecipitated proteins

  • Correlation with mRNA expression levels through RT-PCR

  • Using epitope-tagged recombinant At5g39180 as a positive control

• Genetic validation: Test the antibody in:

  • Knockout/knockdown lines of At5g39180

  • Overexpression lines

  • CRISPR-edited lines with targeted mutations

As noted in comprehensive antibody validation protocols, researchers should document "common pitfalls" such as nonspecific staining patterns due to suboptimal antibody concentration, which may lead to false interpretations of expression patterns .

What protocols are recommended for At5g39180 protein extraction from Arabidopsis tissues?

For optimal extraction of At5g39180 protein from Arabidopsis tissues, the following protocol is recommended based on successful extraction of similar germin-like proteins:

Materials:

• Liquid nitrogen

• Mortar and pestle

• Protein extraction buffer: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 250 mM mannitol, 5 mM EDTA, 10% glycerol, 0.5% (w/v) polyvinylpolypyrollidone, protease inhibitors

• Acid-washed sand

• Miracloth (Calbiochem)

• Ultracentrifuge

Procedure:

• Collect plant tissue (10-day-old seedlings grown under long day conditions work well for germin-like proteins)

• Grind tissue to a powder in liquid nitrogen

• Homogenize frozen powdered tissue with twice the volume of protein extraction buffer plus acid-washed sand

• Filter through Miracloth

• Centrifuge at 15,000 g for 30 min to remove debris

• For membrane-associated fractions, ultracentrifuge the supernatant at 100,000 g for 60 min at 4°C

• Resuspend the pellet in extraction buffer containing 1% Triton X-100

This protocol has been validated for extraction of proteins that interact with BAK1, including potential downstream targets like At5g39180, and ensures preservation of protein activity and native conformation.

How can I use At5g39180 antibody for immunoprecipitation studies to identify interaction partners?

For immunoprecipitation (IP) studies to identify At5g39180 interaction partners, implement the following specialized protocol:

Materials:

• Anti-At5g39180 antibody

• Protein A/G magnetic beads

• Modified extraction buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% IGEPAL CA630, 1× protease inhibitor cocktail, phosphatase inhibitors (2.5 mM Na₃VO₄, 100 nM calyculin A)

• Western blot equipment

Procedure:

• Extract proteins from treated and control plant tissues using the modified extraction buffer (2 mL per gram of tissue)

• Normalize protein concentrations to 1-2 mg/mL using Bradford assay

• Pre-clear lysates with bare beads for 1 hour at 4°C

• Incubate cleared lysates with 2-5 μg of At5g39180 antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-3 hours at 4°C

• Wash beads 3-5 times with binding buffer containing 50 mM Tris-HCl (pH 7.5) and 250 mM NaCl

• Elute proteins with 2× SDS-PAGE sample buffer

• Analyze by SDS-PAGE followed by either:

  • Western blotting for known candidate interactors

  • Silver staining and mass spectrometry for unbiased identification of novel interactors

For validation, perform reciprocal co-immunoprecipitation and include appropriate controls such as IgG control and At5g39180 knockout lines to confirm specificity of the interactions.

How does At5g39180 expression change during plant immune responses and what methods can detect these changes?

At5g39180 expression dynamics during immune responses can be comprehensively monitored using a multi-technique approach:

qRT-PCR analysis:
Implement a time-course experiment following pathogen or PAMP treatment:

• Treat plants with immune elicitors (e.g., flg22, elf18) or pathogens

• Collect samples at multiple timepoints (0, 4, 8, 12, 16, 20, 24, 48 hours)

• Extract RNA using TRIzol or similar reagent

• Synthesize cDNA and perform qRT-PCR with At5g39180-specific primers

• Normalize expression to reference genes (e.g., UBQ5 or β-tubulin)

• Calculate fold changes using the 2^-ΔΔCT method

Protein level detection:

• Perform Western blot analysis using validated At5g39180 antibody

• Compare protein abundance across treatment timepoints

• Quantify band intensity using image analysis software

Research data indicates that At5g39180, as a germin-like protein, is likely to show expression patterns similar to other defense-related genes that are differentially regulated during pathogen-associated molecular pattern (PAMP)-triggered immunity. In BAK1 signaling studies, At5g39180 was down-regulated in BAK1(Y610F) mutant plants, suggesting it may be positively regulated by wild-type BAK1-mediated immunity pathways .

What are the technical challenges in producing specific antibodies against At5g39180 compared to other germin-like proteins?

Producing specific antibodies against At5g39180 presents several technical challenges due to the conserved nature of germin-like proteins:

Epitope selection challenges:

• Germin-like proteins share highly conserved germin domains

• At5g39180 shares sequence homology with other members of the RmlC-like cupins superfamily

• Selection of unique epitopes requires careful bioinformatic analysis to identify regions specific to At5g39180

A strategic approach involves:

• Performing multiple sequence alignment of all Arabidopsis germin-like proteins

• Identifying unique regions with at least 8-10 amino acid differences

• Selecting epitopes from surface-exposed regions while avoiding glycosylation sites

• Using immunoinformatic tools to predict epitope antigenicity and accessibility

Cross-reactivity testing requirements:
Testing must include validation against multiple related proteins. For example, cross-reactivity should be assessed against other germin-like proteins in Arabidopsis, including those encoded by At5g39150 and At5g39120, which are closely related paralogs .

Expression strategy considerations:
Recombinant protein expression for antibody production may require:

• Expression of the full-length protein for polyclonal antibody generation

• Expression of unique epitopes fused to carrier proteins for monoclonal antibody production

• Careful refolding protocols if the protein forms inclusion bodies in E. coli

• Consideration of plant-based expression systems to ensure proper post-translational modifications

How can At5g39180 antibodies be used in protein microarray studies for plant immunity research?

Utilizing At5g39180 antibodies in protein microarray studies requires specialized methodologies:

Production of Arabidopsis protein microarrays:

• Clone At5g39180 cDNA along with other Arabidopsis proteins into Gateway-compatible E. coli expression vectors

• Express and purify RGS-His6-tagged recombinant proteins in high throughput

• Robotically array proteins onto glass slides coated with:

  • Nitrocellulose-based polymer (FAST slides) - detection limit ~2-3.6 fmol per spot

  • Polyacrylamide (PAA slides) - superior sensitivity with detection limit ~0.1-1.8 fmol per spot

Applications in immunity research:

• Antibody specificity profiling:

  • Spot At5g39180 along with other germin-like proteins and control proteins

  • Probe with anti-At5g39180 antibody to assess cross-reactivity

• Protein-protein interaction studies:

  • Probe arrays with fluorescently labeled interacting proteins

  • Detect interaction partners using fluorescence scanning

• Plant immunity pathway mapping:

  • Create arrays containing proteins from immunity pathways

  • Probe with At5g39180 to identify direct interactions

  • Analyze using control antibodies (e.g., anti-RGS-His6) for normalization

As demonstrated in published microarray studies, this approach can successfully detect specific antibody-antigen interactions without cross-reactivity to other spotted proteins, even those from related protein families, making it valuable for confirming antibody specificity .

What are the most effective immunostaining protocols for localizing At5g39180 in plant tissues?

For optimal immunolocalization of At5g39180 in plant tissues, implement the following specialized protocol:

Materials:

• Validated At5g39180-specific antibody

• Fixative: 4% paraformaldehyde in PBS

• Embedding medium: paraffin or LR White resin

• Blocking solution: 5% BSA, 0.1% Tween-20 in PBS

• Fluorescent secondary antibody

• DAPI for nuclear counterstaining

• Anti-fading mounting medium

Tissue preparation:

• Fix freshly harvested tissue in 4% paraformaldehyde for 4 hours at room temperature

• Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

• Embed in paraffin or LR White resin

• Section to 5-10 μm thickness using a microtome

Immunostaining procedure:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval:

  • Heat-induced: 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes

  • Enzymatic: 0.01% trypsin in PBS at 37°C for 10 minutes

• Block in 5% BSA solution for 1 hour at room temperature

• Incubate with primary At5g39180 antibody (1:100-1:500 dilution) overnight at 4°C

• Wash 3× with PBS + 0.1% Tween-20

• Incubate with fluorescent secondary antibody (1:500) for 1-2 hours at room temperature

• Wash 3× with PBS + 0.1% Tween-20

• Counterstain with DAPI (1 μg/mL) for 5 minutes

• Mount in anti-fade medium and seal

Critical controls:

• Primary antibody omission

• Pre-immune serum control

• Peptide competition assay

• Tissue from At5g39180 knockout plants

This protocol is optimized based on successful immunolocalization of similar germin-like proteins in plant tissues, with attention to the secreted nature of At5g39180, which may require careful fixation to preserve extracellular localization .

What are common issues with At5g39180 antibody western blotting and how can they be resolved?

Common Issues and Solutions for At5g39180 Western Blotting:

Recommended Protocol Modifications:

• Extraction buffer optimization:

  • Include 10 mM dithiothreitol to maintain reducing conditions

  • Add 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail

  • For phosphorylation studies, include phosphatase inhibitors (Na₃VO₄, NaF)

• Sample preparation:

  • Heat samples at 70°C for 5 minutes instead of 95°C to prevent aggregation

  • Load 15-20 μg total protein per lane for typical detection

• Transfer optimization:

  • Use wet transfer at 30V overnight at 4°C for efficient transfer of germin-like proteins

  • Verify transfer efficiency with reversible staining (Ponceau S)

These recommendations are based on protocols that have successfully detected similar germin-like proteins in plant extracts .

How can I distinguish between true At5g39180 signal and non-specific binding in my experiments?

To rigorously distinguish between true At5g39180 signal and non-specific binding, implement the following comprehensive validation approach:

1. Genetic validation controls:

• Test the antibody on tissues from:

  • At5g39180 T-DNA insertion mutants (should show no signal)

  • At5g39180 CRISPR knockout lines (should show no signal)

  • At5g39180 overexpression lines (should show enhanced signal)

  • Wild-type plants (baseline signal)

2. Peptide competition assay:

• Pre-incubate the antibody with:

  • Specific peptide used as immunogen (should block specific signal)

  • Unrelated control peptide (should not affect specific signal)

• Compare signal intensity between blocked and unblocked antibody

3. Orthogonal method validation:

• Correlate protein detection with mRNA levels using qRT-PCR

• Compare results with GFP-tagged At5g39180 detection using anti-GFP antibodies

• Validate using mass spectrometry to confirm protein identity in bands recognized by the antibody

4. Cross-reactivity assessment:

• Test antibody against recombinant proteins from related germin-like protein family members

• Create a dot blot array with synthetic peptides representing similar epitopes from related proteins

5. Independent epitope approach:

• Use two antibodies raised against different epitopes of At5g39180

• True signal should be detected by both antibodies at the same molecular weight and location

This systematic approach is based on established antibody validation principles and provides multiple lines of evidence to differentiate specific from non-specific signals .

What are the optimal storage conditions and reconstitution protocols for At5g39180 antibodies to maintain functionality?

For maximum preservation of At5g39180 antibody functionality, implement these storage and handling protocols based on best practices for similar plant protein antibodies:

Storage conditions:

• Long-term storage:

  • Store lyophilized antibody at -20°C in original sealed container

  • Avoid freeze-thaw cycles by storing in smaller aliquots once reconstituted

  • Expected stability: 1+ year for lyophilized form; 6 months for reconstituted form

• Working solution storage:

  • Store at 4°C for up to 2 weeks with preservative (0.02% sodium azide)

  • For longer storage, add 50% glycerol and keep at -20°C

Reconstitution protocol:

• Briefly centrifuge the vial before opening to collect material at the bottom

• Reconstitute in 50 μl sterile water for a typical concentration

• Allow to stand at room temperature for 10 minutes

• Gently mix by inversion and brief vortexing (avoid excessive foaming)

• Make smaller working aliquots (e.g., 10 μl) to avoid repeated freeze-thaw cycles

• Document reconstitution date and conditions

Stability considerations:

• Avoid repeated freeze-thaw cycles (limit to ≤5)

• Monitor antibody performance over time using positive controls

• For experiments requiring maximum sensitivity, use freshly thawed aliquots

These recommendations align with standard protocols for polyclonal antibodies against plant proteins like At5g39180, ensuring maximum retention of specificity and sensitivity over time .

How can computational methods and AI improve At5g39180 antibody design and specificity?

Advanced computational approaches can significantly enhance At5g39180 antibody design through several innovative strategies:

1. Epitope prediction and optimization:

• Implement machine learning algorithms to identify antigenic regions specific to At5g39180

• Use protein structure prediction tools (e.g., AlphaFold) to visualize epitope accessibility

• Apply molecular dynamics simulations to assess epitope flexibility and solvent exposure

2. Direct energy-based preference optimization:

• Leverage conditional diffusion models to jointly model antibody sequences and structures

• Apply residue-level decomposed energy preference to guide generation of antibodies with rational structures

• Implement gradient surgery to address conflicts between various types of energy (attraction vs. repulsion)

3. Antibody-antigen binding simulation:

• Use molecular docking to predict binding modes between candidate antibodies and At5g39180

• Calculate binding energies to rank antibody candidates

• Perform in silico mutagenesis to optimize binding interface residues

4. Cross-reactivity prediction:

• Apply sequence and structural alignment tools to identify potentially cross-reactive proteins

• Calculate similarity scores between epitopes of At5g39180 and other germin-like proteins

• Design computational filters to exclude antibody candidates with potential cross-reactivity

Research has demonstrated that generative AI approaches can achieve binding rates of 10.6% for heavy chain CDR3 designs in zero-shot antibody design efforts , suggesting similar approaches could be applied to develop highly specific At5g39180 antibodies with reduced experimental screening requirements.

What are the emerging applications of At5g39180 antibodies in plant-pathogen interaction studies?

At5g39180 antibodies are becoming valuable tools in advanced plant-pathogen interaction studies through several innovative applications:

1. Spatiotemporal mapping of defense responses:

• Monitor At5g39180 protein localization before and after pathogen infection

• Track protein redistribution during immune responses using immunofluorescence microscopy

• Correlate protein accumulation with infection progression in different tissue types

2. Identification of post-translational modifications:

• Develop modification-specific antibodies (phospho-, glyco-, ubiquitin-specific)

• Map PTM changes during infection using Western blot analysis

• Correlate modifications with protein activity and interaction patterns

3. Proximity-dependent labeling:

• Use antibody-enzyme conjugates (e.g., APEX, BioID) to identify proteins in close proximity to At5g39180 during immune responses

• Map the dynamic interactome changes upon pathogen challenge

• Identify novel components of immunity pathways

4. MAPK activation monitoring:

• Develop assays linking At5g39180 to MAPK cascade activation

• Compare phosphorylation patterns with standardized immune elicitation assays

• Examine activation in response to flg22 and elf18 treatments (100 nM for 5 min)

5. Integration with split-root experiments:

• Use At5g39180 antibodies to study systemic acquired resistance signaling

• Monitor protein abundance in infected versus uninfected portions of split-root systems

• Correlate with phytohormone measurements (SA, JA, JA-Ile)

These approaches are particularly valuable for understanding how germin-like proteins like At5g39180 contribute to PAMP-triggered immunity and interact with BAK1-dependent defense signaling pathways.

How can At5g39180 antibodies be used to study protein-protein interactions in plant immune signaling networks?

At5g39180 antibodies can be leveraged for comprehensive mapping of protein-protein interactions in plant immune networks through these advanced methodologies:

1. Co-immunoprecipitation coupled with mass spectrometry:

• Use At5g39180 antibodies to pull down the protein and its interacting partners

• Analyze protein complexes by LC-MS/MS to identify novel interactors

• Compare interactome composition before and after immune elicitation

• Validate key interactions using reciprocal co-IP and BiFC

2. Proximity-dependent labeling:

• Generate fusion proteins combining At5g39180 with BioID or APEX2

• Use antibodies to immunoprecipitate At5g39180 complexes after biotin labeling

• Identify labeled proteins that exist in close proximity (within ~10 nm)

• Map spatial organization of At5g39180 within defense-related protein complexes

3. In situ protein-protein interaction analysis:

• Apply proximity ligation assay (PLA) using At5g39180 antibodies with antibodies against candidate interactors

• Visualize interaction events as fluorescent dots in fixed cells/tissues

• Quantify changes in interaction frequency during immune responses

4. Integrative network analysis:

• Combine antibody-based interaction data with transcriptomic data

• Correlate At5g39180 expression patterns with interacting partners

• Map into known defense signaling pathways (e.g., BAK1-mediated PTI signaling)

Implementation example: Based on research showing At5g39180 is down-regulated in BAK1(Y610F) plants , investigators could use At5g39180 antibodies to test for direct interaction with BAK1 or downstream MAPK components, potentially revealing how this germin-like protein contributes to PAMP-triggered immunity signaling cascades.

What quality control measures should be implemented when validating At5g39180 antibody batches for consistency across experiments?

To ensure experimental reproducibility with At5g39180 antibodies, implement this comprehensive quality control framework:

1. Standardized validation panel for each batch:

2. Reference standard development:

• Create a stable positive control (e.g., recombinant At5g39180)

• Establish a standard curve for each antibody batch

• Document batch-specific optimal working dilutions

3. Application-specific validation:

• For Western blot: Test linearity across protein concentration range

• For immunoprecipitation: Verify % recovery of spiked protein

• For immunolocalization: Confirm reproducible staining patterns

4. Stability monitoring program:

• Test antibody performance at defined intervals (0, 3, 6, 12 months)

• Document any sensitivity loss over time

• Establish maximum shelf-life based on performance metrics

5. Documentation requirements: