At1g59780 Antibody

What is At1g59780 and why is it important in plant immunity research?

At1g59780 is a putative disease resistance protein in Arabidopsis that belongs to the nucleotide-binding leucine-rich repeat (NB-LRR) class of plant resistance (R) proteins. This protein plays a critical role in plant immunity by recognizing pathogen effectors and triggering defense responses.

The gene is part of the disease resistance gene family similar to SNC1 (Suppressor of NPR1, Constitutive 1), which functions in plant immune responses. Research indicates that At1g59780 contributes to pathogen recognition and subsequent defense signaling cascades . Understanding its function is crucial for developing strategies to enhance plant disease resistance in agricultural applications.

What types of antibodies are typically used for studying disease resistance proteins like At1g59780?

Several antibody types are employed in At1g59780 research:

• Polyclonal antibodies: Generated by immunizing animals (typically rabbits) with peptides or recombinant proteins derived from At1g59780. These recognize multiple epitopes and provide high sensitivity, though specificity may vary between batches.

• Monoclonal antibodies: Produced from single B-cell clones, offering high specificity to individual epitopes on At1g59780, with greater consistency between batches.

• Custom antibodies: For At1g59780 research, custom antibodies can be developed against specific protein domains, phosphorylation sites, or protein variants .

An example from related research shows that anti-SNC1 polyclonal antibodies produced in rabbits have been successfully used to detect disease resistance proteins in Arabidopsis .

How should At1g59780 antibody validation be performed for plant research applications?

Comprehensive validation should include:

Western blot analysis:

• Test antibody against wild-type plant tissue and knockout/knockdown lines

• Verify protein size matches predicted molecular weight (~48-70 kDa, depending on modifications)

• Include positive controls (e.g., plants overexpressing At1g59780)

• Test for cross-reactivity with related R-proteins

Specificity testing:

• Peptide competition assays to verify epitope specificity

• Immunoprecipitation followed by mass spectrometry

• Testing across tissue types and developmental stages

Controls for validation:

• Use T-DNA insertional mutants lacking At1g59780 expression as negative controls

• Include overexpression lines as positive controls

• Employ multiple antibody dilutions to optimize signal-to-noise ratio

A validation approach similar to that used for ACBP6-specific antibodies in Arabidopsis could be applied, where antibody specificity was confirmed using both wild-type and knockout lines .

What expression patterns does At1g59780 typically show during plant immune responses?

At1g59780 expression typically follows patterns similar to other disease resistance proteins:

• Basal expression: Low to moderate levels in healthy plants

• Pathogen-induced expression: Significant upregulation 24-48 hours after pathogen challenge

• Tissue specificity: Primarily expressed in leaf tissue, with lower expression in stems and roots

• Developmental regulation: Expression varies across developmental stages

Studies of similar resistance proteins have shown that protein abundance can be assessed via western blot analysis using specific antibodies. For example, research on SNC1 demonstrated that protein levels increased significantly upon pathogen challenge, with nuclear accumulation being particularly important for defense activation .

How can subcellular localization of At1g59780 be determined using antibody-based techniques?

Several methodologies enable accurate subcellular localization:

Immunofluorescence microscopy:

• Fix plant tissues with paraformaldehyde (4%)

• Perform cell wall digestion with cellulase/macerozyme

• Block with BSA (3-5%) to prevent non-specific binding

• Incubate with At1g59780 primary antibody (1:100-1:500 dilution)

• Apply fluorophore-conjugated secondary antibody

• Counterstain with organelle markers (e.g., DAPI for nuclei)

Cell fractionation with immunoblotting:

• Fractionate plant tissues into cytosolic, nuclear, membrane, and organelle fractions

• Perform western blotting on each fraction

• Probe with At1g59780 antibody

• Use fraction-specific markers as controls:

  • GAPDH for cytosolic fraction

  • Histone H3 for nuclear fraction

  • Membrane proteins (e.g., H⁺-ATPase) for membrane fraction

This approach has been successfully used for ACBP6 localization in Arabidopsis, confirming its cytosolic location through differential centrifugation followed by western-blot analysis using specific antibodies .

What considerations are important when studying protein-protein interactions of At1g59780?

When investigating At1g59780 interaction partners:

Co-immunoprecipitation (Co-IP):

• Use crosslinking agents (0.5-1% formaldehyde) to stabilize transient interactions

• Optimize extraction buffers for plant proteins (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40)

• Include protease inhibitors to prevent degradation

• Use magnetic beads conjugated with At1g59780 antibody

• Validate interactions through reciprocal Co-IP

• Confirm with mass spectrometry analysis

Controls for interaction studies:

• IgG control precipitation

• Knockout/knockdown lines

• Competition with peptides

• Denaturing conditions to confirm specificity

Studies with similar resistance proteins have shown that interactions with transcriptional corepressors, like TPR1, are critical for defense activation, suggesting At1g59780 might have similar interacting partners .

How can phosphorylation status of At1g59780 be detected using antibody-based methods?

Phosphorylation detection strategies include:

Phospho-specific antibodies:

• Develop antibodies against predicted phosphorylation sites in At1g59780

• Validate using phosphatase treatment controls

• Compare signals before and after pathogen challenge

Phos-tag SDS-PAGE with immunoblotting:

• Incorporate Phos-tag reagent (50-100 μM) and MnCl₂ in acrylamide gels

• Separate proteins based on phosphorylation status

• Detect with At1g59780 antibody

• Quantify band shifts corresponding to phosphorylated forms

Mass spectrometry validation:

• Immunoprecipitate At1g59780 protein

• Digest with trypsin

• Analyze phosphopeptides using LC-MS/MS

• Compare phosphorylation patterns before/after immune activation

This approach is similar to techniques used for other kinase-regulated proteins, where phospho-specific antibodies can detect activation states, as demonstrated in the AKT1 (pSer473) antibody studies .

What approaches can distinguish between closely related R-proteins when using At1g59780 antibodies?

Distinguishing between related R-proteins requires:

Epitope selection strategies:

• Target unique regions outside conserved NB-LRR domains

• Use C-terminal specific antibodies (highest variability region)

• Develop peptide-specific antibodies against unique sequences

Analytical approaches:

• Pre-absorb antibodies with recombinant related proteins

• Use knockout lines as negative controls

• Perform immunoprecipitation followed by mass spectrometry

• Employ competitive ELISA with related peptides

Bioinformatic analysis:

Research on plant disease resistance proteins indicates that antibody cross-reactivity is a significant challenge, requiring careful validation strategies similar to those used in studies of MAC207 antibodies for plant proteins .

What protocols are recommended for western blot analysis using At1g59780 antibodies?

Optimized western blot protocol for At1g59780 detection:

Sample preparation:

• Grind 100 mg plant tissue in liquid nitrogen

• Extract proteins in buffer containing:

  • 50 mM Tris-HCl pH 7.5

  • 150 mM NaCl

  • 10% glycerol

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Centrifuge at 15,000 × g for 15 min at 4°C

• Quantify protein concentration using Bradford assay

Western blot procedure:

• Separate 10-30 μg protein on 10% SDS-PAGE

• Transfer to PVDF membrane (100V for 60 min)

• Block with 5% non-fat milk in TBST for 1 hour

• Incubate with At1g59780 primary antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST (10 min each)

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour

• Wash 3× with TBST

• Develop using ECL substrate and detect with chemiluminescence imager

Critical controls:

• Use GAPDH or actin as loading controls

• Include recombinant At1g59780 protein as positive control

• Include samples from knockout lines as negative controls

This protocol incorporates methods successfully used in studies examining ACBP6 protein expression in Arabidopsis, which showed increased protein accumulation after cold treatment .

How can immunoprecipitation be optimized for studying At1g59780 and its binding partners?

Optimized immunoprecipitation protocol:

Sample preparation:

• Harvest 5 g plant tissue and crosslink with 1% formaldehyde for 10 min

• Quench with 125 mM glycine

• Grind tissue in liquid nitrogen

• Extract in IP buffer:

  • 50 mM HEPES pH 7.5

  • 150 mM NaCl

  • 10% glycerol

  • 1 mM EDTA

  • 0.5% NP-40

  • Protease/phosphatase inhibitors

• Sonicate briefly (3 × 10 sec, 30% amplitude)

• Centrifuge at 15,000 × g for 15 min at 4°C

Immunoprecipitation:

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Incubate cleared lysate with 2-5 μg At1g59780 antibody overnight at 4°C

• Add 50 μl Protein A/G magnetic beads for 2 hours

• Wash beads 4× with IP buffer

• Elute proteins with 50 μl 2× SDS sample buffer at 95°C for 5 min

Analysis options:

• Western blot for known interactors

• Silver staining followed by mass spectrometry

• Targeted PCR for associated nucleic acids

This approach builds on techniques used in plant immunity research, where immunoprecipitation has been used to identify protein complexes involved in defense signaling .

What methods are effective for quantifying At1g59780 protein expression during plant immune responses?

Several quantification methods provide reliable results:

Quantitative western blotting:

• Use a dilution series of recombinant At1g59780 protein to create a standard curve

• Load equal amounts of total protein from samples

• Include internal standards (e.g., recombinant protein spiked into plant extracts)

• Analyze band intensities using software (ImageJ)

• Normalize to reference proteins (GAPDH, actin, tubulin)

ELISA:

• Develop sandwich ELISA using two antibodies recognizing different At1g59780 epitopes

• Create standard curves using purified recombinant protein

• Analyze samples in technical triplicates

• Calculate protein concentration from 4-parameter logistic curves

Flow cytometry (for single-cell analysis):

• Isolate protoplasts from plant tissues

• Fix and permeabilize cells

• Stain with fluorescently-labeled At1g59780 antibody

• Analyze using flow cytometer

• Quantify based on fluorescence intensity

Based on similar research, western blot analysis has been effective for quantifying protein expression changes, as demonstrated in studies where ACBP6 protein showed highest accumulation at 48 hours following cold treatment .

What controls should be included when using At1g59780 antibodies in immunofluorescence studies?

Essential controls for immunofluorescence:

Experimental controls:

• Primary antibody controls:

  • Omit primary antibody (secondary antibody only)

  • Use non-immune serum from same species

  • Pre-absorb antibody with immunizing peptide

• Sample controls:

  • At1g59780 knockout/knockdown plants

  • At1g59780 overexpression lines

  • Wild-type untreated plants vs. pathogen-challenged plants

• Specificity controls:

  • Competitive blocking with immunizing peptide

  • Testing antibody across related plant species

  • Dual labeling with antibodies against known interactors

• Technical controls:

  • Autofluorescence quenching

  • Counterstains for specific organelles (DAPI for nuclei, MitoTracker for mitochondria)

  • Z-stack imaging to confirm localization patterns

The approach used in ACBP6-GFP localization studies provides a useful model, where both anti-GFP and anti-ACBP6 antibodies were used to confirm subcellular localization in transgenic Arabidopsis seedlings .

What are common causes of non-specific binding with At1g59780 antibodies and how can they be addressed?

Common causes and solutions for non-specific binding:

Causes of non-specific binding:

• Antibody cross-reactivity with related R-proteins

• Insufficient blocking

• Overly concentrated primary antibody

• Sample overloading

• Excessive secondary antibody

Solutions:

• Optimization of blocking conditions:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Increase blocking time (2-3 hours or overnight)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Test serial dilutions (1:500, 1:1000, 1:2000, 1:5000)

  • Reduce incubation time or temperature

  • Use fresh antibody aliquots to avoid freeze-thaw cycles

• Additional washing steps:

  • Increase number of washes (5-6 times)

  • Extend washing duration (15-20 minutes per wash)

  • Use higher salt concentration in wash buffer (up to 500 mM NaCl)

• Pre-absorption strategies:

  • Pre-incubate antibody with plant extract from knockout lines

  • Use peptide competition to verify specific signals

Based on antibody validation studies, pre-absorption with related proteins can significantly reduce non-specific binding, as demonstrated in research on plant-specific antibodies .

How can sensitivity be improved when detecting low-abundance proteins like At1g59780?

Strategies to enhance detection sensitivity:

Sample preparation enhancements:

• Enrich target protein via immunoprecipitation before western blotting

• Fractionate cellular components to concentrate the protein of interest

• Use plant tissue with highest At1g59780 expression (e.g., young leaves after pathogen treatment)

• Treat plants to induce protein expression (e.g., pathogen-associated molecular patterns)

Detection system improvements:

• Use high-sensitivity ECL substrates for western blots

• Employ signal amplification systems:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

• Switch to fluorescent secondary antibodies and use laser-based scanners

• Consider using quantum dots for increased sensitivity in immunofluorescence

Protocol modifications:

• Extend primary antibody incubation (overnight at 4°C)

• Use signal enhancers in blocking buffer

• Reduce washing stringency slightly without compromising specificity

• Load maximum possible protein amount without causing lane distortion

Similar approaches have been used to detect low-abundance proteins in plant immunity studies, where detection of disease resistance proteins required specialized extraction and detection methods .

How can batch-to-batch variability in At1g59780 antibody performance be managed?

Strategies to minimize variability impacts:

Antibody management practices:

• Purchase larger antibody lots when possible

• Aliquot new antibodies into single-use volumes

• Store according to manufacturer recommendations (typically -20°C or -80°C)

• Track lot numbers and validate each new lot

Standardization approaches:

• Create standard curves with recombinant protein for each experiment

• Include consistent positive controls across experiments

• Normalize to internal reference proteins

• Use the same protocol conditions across experiments

Documentation and validation:

• Document antibody performance metrics for each lot:

  • Working dilution

  • Signal-to-noise ratio

  • Detection limit

• Perform side-by-side comparisons between old and new lots

• Consider using monoclonal antibodies when available for greater consistency

Alternative approaches:

• Maintain parallel detection methods (e.g., tagged protein systems)

• Consider developing a monoclonal antibody for critical applications

Finite mixture models can be helpful for analyzing antibody data with batch-to-batch variability, as demonstrated in serological data analysis for distinguishing antibody-positive and antibody-negative results .

What approaches can help distinguish At1g59780 from other related R-proteins in complex samples?

Advanced techniques for discriminating between related proteins:

Analytical approaches:

• 2D immunoblotting:

  • Separate proteins first by isoelectric point, then by molecular weight

  • Detect with At1g59780 antibody

  • Compare migration patterns with predicted values

• Sequential immunoprecipitation:

  • Deplete related proteins using specific antibodies

  • Then immunoprecipitate At1g59780

  • Analyze by western blot or mass spectrometry

• Competitive binding assays:

  • Pre-incubate antibody with increasing concentrations of related protein peptides

  • Measure reduction in signal to quantify cross-reactivity

Genetic approaches:

• Use multiple plant genotypes:

  • Wild-type

  • At1g59780 knockouts

  • Knockouts of related R-genes

  • Double/triple mutants

• Generate epitope-tagged versions for unambiguous detection

Computational analysis:

• Apply finite mixture models to antibody binding data to separate signals from closely related proteins

• Use machine learning algorithms to distinguish binding patterns

Studies with MAC207 antibodies showed that epitope competition assays can effectively determine antibody specificity, with the most effective oligosaccharide competitors helping to distinguish between related plant proteins .