At4g19865 Antibody

What is the At4g19865 protein and why is it significant for antibody development?

The At4g19865 gene encodes an F-box/Kelch-repeat protein in Arabidopsis thaliana with the peptide sequence K.IEFGNVNEMCAYDTKLCK.W identified in proteomic studies . F-box proteins typically function as components of SCF ubiquitin ligase complexes that regulate protein degradation pathways, while Kelch-repeat domains facilitate protein-protein interactions. The protein appears to undergo post-translational modifications, including tyrosine nitration, as detected in Arabidopsis thaliana protein extracts using immunoprecipitation techniques with anti-3-nitroY antibodies .

Developing antibodies against At4g19865 is significant because it would enable researchers to study its expression patterns, subcellular localization, interaction partners, and post-translational modifications under various experimental conditions. These studies could reveal critical insights into plant stress responses and developmental processes regulated by this protein. Additionally, such antibodies would help elucidate the protein's role in ubiquitin-mediated protein degradation pathways and the functional significance of its tyrosine nitration.

What types of antibodies can be developed against the At4g19865 protein?

Several types of antibodies can be developed against At4g19865, each with distinct advantages for different research applications:

• Polyclonal antibodies: Generated by immunizing animals (typically rabbits, goats, or chickens) with purified At4g19865 protein or synthesized peptides from unique regions of the protein. These antibodies recognize multiple epitopes, providing robust detection but potentially variable specificity between batches.

• Monoclonal antibodies: Produced from single B-cell clones after immunization, these antibodies recognize specific epitopes with high consistency across experiments. Their development follows approaches similar to the monoclonal antibody generation procedure used for the anti-Rhamnogalacturonan I antibody described in search result , where specific clone selection (e.g., "7H4.B6.F6") ensures reproducibility.

• Recombinant antibodies: Engineered antibody fragments (Fab, scFv) expressed in bacterial or mammalian systems, offering consistent production without animal immunization.

• Post-translational modification-specific antibodies: Specialized antibodies that specifically recognize nitrated forms of At4g19865, similar to the anti-3-nitroY antibodies used to detect protein nitration in Arabidopsis . These would distinguish between modified and unmodified forms of the protein.

The choice depends on research goals, available resources, and intended applications, with monoclonal antibodies typically preferred for quantitative studies requiring high specificity.

What are the key considerations for validating an At4g19865 antibody?

Rigorous validation of antibodies against At4g19865 requires multiple complementary approaches:

• Western blot specificity testing: The antibody should detect a band of the expected molecular weight (~40 kDa based on reference ) in wild-type Arabidopsis extracts that disappears or is significantly reduced in At4g19865 knockout lines. Specificity can be further confirmed using on-membrane reduction techniques similar to those used for validating anti-3-nitroY antibodies .

• Peptide competition assays: Pre-incubating the antibody with the synthetic peptide used for immunization should abolish or significantly reduce signal if the antibody is specific.

• Immunoprecipitation followed by mass spectrometry: The antibody should pull down At4g19865 from plant extracts, which can be verified by mass spectrometry analysis of the immunoprecipitated fraction. This approach mirrors the methodology used to identify nitrated proteins in Arabidopsis .

• Cross-reactivity assessment: Testing against related F-box/Kelch-repeat proteins in Arabidopsis to ensure the antibody doesn't recognize similar proteins.

• Immunolocalization controls: For antibodies intended for microscopy applications, signal should be absent in knockout lines and present in expected subcellular locations in wild-type plants.

These validation steps establish the reliability and specificity of the antibody before its application in experimental research, preventing misinterpretation of results due to non-specific binding.

What extraction conditions are optimal for detecting At4g19865 protein in plant tissues?

Optimizing extraction conditions for At4g19865 detection requires consideration of protein characteristics and post-translational modifications:

• Buffer composition:

  • Tris-HCl (50 mM, pH 7.5-8.0) provides appropriate pH stability

  • NaCl (150-300 mM) maintains protein solubility while reducing non-specific interactions

  • EDTA (1-5 mM) prevents metal-dependent proteolysis

  • Glycerol (5-10%) enhances protein stability

  • Non-ionic detergents (0.1-1% Triton X-100 or NP-40) solubilize membrane-associated fractions

• Protease inhibitors:

  • Complete protease inhibitor cocktail to prevent degradation

  • Specific inhibitors for plant proteases (e.g., PMSF, leupeptin, pepstatin A)

  • Proteasome inhibitors (MG132, 10-50 μM) particularly important as At4g19865 is an F-box protein likely regulated by the ubiquitin-proteasome system

• Modification-preserving additions:

  • Phosphatase inhibitors (sodium fluoride, sodium orthovanadate)

  • Deubiquitinase inhibitors (N-ethylmaleimide)

  • Nitration-preserving conditions (avoid reducing agents that may reduce nitrotyrosine)

• Extraction procedure:

  • Flash-freezing tissue in liquid nitrogen followed by grinding to fine powder

  • Maintaining cold temperature throughout extraction (4°C)

  • Clearing extracts by centrifugation (14,000 × g, 15 minutes)

  • Filtration through 0.45 μm filters for particularly complex samples

These conditions should be optimized based on the specific tissue type and experimental goals, with particular attention to preserving nitrated forms if studying post-translational modifications identified in proteomic studies .

How can I use antibodies to study post-translational modifications of At4g19865 protein?

Studying post-translational modifications of At4g19865, particularly tyrosine nitration, requires specialized immunological approaches:

• Dual antibody strategy: Use both general anti-At4g19865 antibodies and modification-specific antibodies (such as anti-3-nitroY) to compare total protein versus modified forms. This approach was successfully used to detect nitrated proteins in Arabidopsis .

• Sequential immunoprecipitation: First immunoprecipitate with anti-At4g19865 antibodies, then probe the precipitated material with anti-3-nitroY antibodies (or vice versa). This confirms that the specific protein of interest is modified rather than co-precipitating proteins.

• Mass spectrometry validation: After immunoprecipitation, analyze the purified protein by mass spectrometry to identify modified residues. For nitrotyrosine, look for the characteristic +45 Da mass shift seen in other nitrated Arabidopsis proteins like Rubisco activase (showing a +45.07 shift as documented in the mass spectrometry data table) .

• Site-directed mutagenesis studies: Generate Arabidopsis plants expressing At4g19865 with mutations in specific tyrosine residues predicted to be nitration sites, then use antibodies to study how these mutations affect protein function, localization, or stability.

• Comparative analysis under stress conditions: Compare nitration patterns under various oxidative and nitrosative stress conditions known to induce protein nitration, establishing correlation between the modification and particular physiological states.

These approaches provide insights into how post-translational modifications regulate At4g19865 function and may reveal novel regulatory mechanisms in plant stress responses.

What are the best approaches for studying At4g19865 protein-protein interactions using antibodies?

Investigating protein-protein interactions involving At4g19865 can employ several complementary antibody-based techniques:

• Co-immunoprecipitation (Co-IP): Use anti-At4g19865 antibodies to pull down the protein along with its interaction partners from plant extracts. The technique can be optimized similar to the immunoprecipitation approach used for nitrated proteins in Arabidopsis , but with conditions preserving native protein complexes. Precipitated complexes can then be analyzed by mass spectrometry or western blotting with antibodies against suspected interaction partners.

• Proximity ligation assay (PLA): This in situ technique uses pairs of antibodies (anti-At4g19865 and antibodies against potential interaction partners) followed by oligonucleotide-conjugated secondary antibodies that enable signal amplification when proteins are in close proximity (<40 nm). This provides spatial information about where interactions occur in plant tissues.

• ChIP-seq for chromatin associations: If At4g19865 associates with chromatin (directly or indirectly), chromatin immunoprecipitation using anti-At4g19865 antibodies followed by sequencing can identify genomic regions where these interactions occur.

• Immunofluorescence co-localization: Using fluorescently labeled antibodies against At4g19865 and suspected interaction partners to determine whether they co-localize in plant cells, providing indirect evidence of potential interactions.

• Bimolecular fluorescence complementation (BiFC) with antibody validation: While BiFC itself uses fluorescent protein fragments, antibodies can validate the expression levels of the fusion proteins used in BiFC experiments, ensuring reliable interpretation of results.

These techniques reveal the composition of protein complexes containing At4g19865 and provide insights into its biological functions in ubiquitin-mediated protein degradation pathways.

How can epitope mapping improve the specificity of At4g19865 antibodies?

Epitope mapping can significantly enhance antibody specificity for At4g19865 research:

• Peptide array analysis: Synthesize overlapping peptides spanning the entire At4g19865 sequence and test antibody binding to identify specific epitope regions. This approach, similar to epitope characterization methods used for other plant antibodies , can identify the precise amino acid sequence recognized by the antibody.

• Structural considerations: Use protein structure prediction tools to identify surface-exposed regions of At4g19865 that are likely to be good epitopes and design antibodies targeting these regions. For F-box/Kelch-repeat proteins, the kelch repeats typically form a β-propeller structure with exposed loops that make good antibody targets.

• Comparative sequence analysis: Compare At4g19865 sequence with related Arabidopsis F-box proteins to identify unique regions suitable for raising highly specific antibodies. This minimizes cross-reactivity with related proteins.

• Recombinant fragment approach: Express different domains of At4g19865 (F-box domain, Kelch-repeat domain) separately and determine which fragments are recognized by the antibody.

• Cross-reactivity testing: Once epitopes are identified, test antibody reactivity against closely related F-box/Kelch-repeat proteins to ensure specificity for At4g19865.

This information guides the development of more specific antibodies or the refinement of existing ones, similar to the approach that resulted in highly specific monoclonal antibodies like the anti-Rhamnogalacturonan I antibody described in search result .

What strategies can be used to study At4g19865 protein dynamics during plant development and stress responses?

Studying At4g19865 protein dynamics across different conditions requires sophisticated antibody-based approaches:

• Quantitative western blotting: Use anti-At4g19865 antibodies with appropriate loading controls to quantify protein levels across developmental stages or stress treatments. This requires establishing the linear range of detection and appropriate normalization methods.

• Tissue-specific immunohistochemistry: Apply anti-At4g19865 antibodies to tissue sections from plants at different developmental stages or under various stress conditions to visualize changes in protein localization and abundance. This provides spatial context to expression pattern changes.

• Immunoprecipitation coupled with pulse-chase analysis: Label newly synthesized proteins with a radioactive or chemical tag, then use antibodies to immunoprecipitate At4g19865 at different time points to measure protein turnover rates under different conditions.

• Modification-specific antibody comparison: Use both general anti-At4g19865 antibodies and nitrotyrosine-specific antibodies to monitor changes in the ratio of modified to unmodified protein, similar to approaches used to study nitrated proteins in Arabidopsis .

• Multiplexed immunoassays: Develop antibody-based arrays or multiplexed detection systems to simultaneously monitor At4g19865 along with other related proteins in response to developmental or environmental signals.

These approaches reveal how At4g19865 protein levels, localization, and modifications change in response to developmental signals or environmental stresses, providing insights into its physiological roles.

How can I develop an ELISA assay for quantifying At4g19865 protein levels?

Developing an ELISA for At4g19865 protein quantification involves these methodological steps:

• Antibody pair selection: For sandwich ELISA, use two antibodies recognizing different epitopes on At4g19865 - one for capture and one for detection. If only one antibody is available, develop a competitive ELISA format where sample At4g19865 competes with a standard amount of labeled protein for antibody binding.

• Assay format optimization:

  • Direct ELISA: Coat plates with plant extract, detect with anti-At4g19865 antibody

  • Sandwich ELISA: Coat with capture antibody, add sample, detect with a second antibody

  • Competitive ELISA: Compete sample At4g19865 with a standard amount of purified protein

• Standard curve preparation: Generate recombinant At4g19865 protein for creating standard curves. The curve fitting should follow standard models similar to those used in other immunoassays, such as the four-parameter logistic curve model: y = (A - D)/( 1 + (x/C)^B ) + D, as demonstrated in other antibody-based quantification systems .

• Protocol optimization:

  • Coating buffer composition and pH (typically carbonate/bicarbonate buffer pH 9.6)

  • Blocking solution (typically BSA or casein-based)

  • Sample preparation method (extraction buffer, dilution factor)

  • Antibody concentrations and incubation times

  • Washing protocols to minimize background

• Validation:

  • Test specificity using extracts from At4g19865 knockout plants

  • Determine lower limit of detection and quantification

  • Assess linear range of measurement

  • Evaluate reproducibility (intra- and inter-assay variation)

This methodical approach yields a reliable assay for quantifying At4g19865 protein levels across various experimental conditions, suitable for monitoring changes during development or stress responses.

What considerations are important when using At4g19865 antibodies for immunohistochemistry?

Successful immunohistochemical detection of At4g19865 in plant tissues requires optimization of several key parameters:

• Tissue fixation and processing:

  • Paraformaldehyde fixation (typically 4%) preserves protein structure while maintaining antigenic sites

  • Alternative fixatives (Carnoy's, glutaraldehyde) may be tested if standard fixation fails

  • Embedding medium selection (paraffin, resin, cryosectioning) affects epitope accessibility

  • Section thickness optimization (typically 5-10 μm for light microscopy)

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K, trypsin) for fixed epitopes

  • Optimization of retrieval duration and temperature

  • Testing multiple retrieval methods to determine optimal conditions

• Blocking and antibody incubation:

  • Blocking with appropriate sera (5-10% normal serum from secondary antibody host species)

  • Adding blocking agents to reduce non-specific binding (BSA, casein, commercial blockers)

  • Primary antibody dilution optimization (typically 1:100-1:1000)

  • Incubation conditions (time, temperature, humidity)

• Detection system selection:

  • Fluorescent secondary antibodies for co-localization studies

  • Enzymatic detection (HRP, AP) with appropriate substrates for permanent slides

  • Signal amplification systems (tyramide, avidin-biotin) for low-abundance proteins

  • Counterstains for tissue architecture visualization

• Controls:

  • Negative controls (no primary antibody, isotype control, pre-immune serum)

  • Positive controls (tissues known to express At4g19865)

  • Peptide competition controls to confirm specificity

  • Tissues from At4g19865 knockout plants as definitive negative controls

These methodological considerations ensure specific and reproducible detection of At4g19865 in plant tissues, enabling studies of its expression patterns and subcellular localization.

What approaches can resolve contradictory results when using At4g19865 antibodies?

When faced with contradictory antibody-based results for At4g19865, apply these systematic troubleshooting approaches:

• Antibody validation reassessment:

  • Re-test antibody specificity using western blots on wild-type vs. knockout extracts

  • Perform peptide competition assays to confirm epitope specificity

  • Test multiple antibodies targeting different epitopes of At4g19865

  • Verify antibody batch consistency and storage conditions

• Experimental condition variations:

  • Test different protein extraction methods (native vs. denaturing conditions)

  • Optimize sample preparation (reducing vs. non-reducing conditions)

  • Try different blocking reagents (BSA vs. milk vs. commercial blockers)

  • Compare detection systems (chemiluminescence vs. fluorescence)

• Post-translational modification considerations:

  • Test for conditions that may induce modifications affecting antibody recognition

  • Use modification-specific antibodies (like anti-3-nitroY) alongside total protein antibodies

  • Apply treatments that remove specific modifications (phosphatases, sodium dithionite for nitrotyrosine reduction as used in validation of anti-3-nitroY antibodies)

• Alternative validation approaches:

  • Complement antibody-based methods with non-antibody techniques

  • Use mass spectrometry for protein identification

  • Employ genetic approaches (reporter gene fusions)

  • Implement on-membrane reduction of 3-nitroY to 3-aminoY with sodium dithionite as a specificity control, as demonstrated for nitrated proteins in Arabidopsis

• Systematic documentation:

  • Document all experimental conditions thoroughly

  • Compare results across different plant growth stages and conditions

  • Collaborate with other labs to independently verify findings

  • Implement blind sample coding to eliminate unconscious bias

How can I optimize immunoprecipitation of At4g19865 for downstream mass spectrometry analysis?

Optimizing immunoprecipitation of At4g19865 for mass spectrometry requires specific considerations:

• Extraction buffer optimization:

  • Use buffers compatible with mass spectrometry (avoid detergents like SDS, NP-40)

  • Consider MS-compatible detergents (Rapigest, ProteaseMAX)

  • Include protease inhibitors without polyethylene glycol contamination

  • Maintain protein modifications with appropriate inhibitors (phosphatase inhibitors, deubiquitinase inhibitors)

• Antibody immobilization strategy:

  • Covalent coupling to beads to prevent antibody contamination in eluate

  • Use zero-length cross-linkers that don't introduce chemical modifications

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Consider using magnetic beads for gentle recovery with minimal background

• Washing protocols:

  • Develop stringent washing steps to remove non-specific binders

  • Use MS-compatible buffers for final washes

  • Perform sufficient wash steps without disrupting specific interactions

  • Include salt gradients in wash buffers to discriminate between high and low-affinity interactions

• Elution methods:

  • Peptide elution for antibodies raised against specific peptides

  • Acid elution (glycine pH 2.5) with immediate neutralization

  • On-bead digestion to avoid contamination from antibody elution

  • Filter-aided sample preparation (FASP) for compatibility with MS

• Controls and validation:

  • Include IgG control immunoprecipitations

  • Perform parallel IP-western blot to confirm successful pulldown

  • Include spike-in controls to assess recovery efficiency

  • Validate mass spectrometry identification with independent methods

This approach enables identification of At4g19865 post-translational modifications and interaction partners with high confidence, similar to the methods used to identify nitrated proteins in Arabidopsis thaliana .

How can At4g19865 antibodies be used to study protein nitration in plant stress responses?

Using At4g19865 antibodies to study protein nitration during stress requires specialized methodological approaches:

• Dual immunodetection workflow:

  • Use anti-At4g19865 antibodies to track total protein levels

  • Use anti-3-nitroY antibodies to detect nitrated forms, employing the validation approaches demonstrated in Arabidopsis nitration studies (including sodium dithionite reduction of 3-nitroY to 3-aminoY as a control)

  • Compare ratios of nitrated to total protein across stress conditions

• Stress treatment experimental design:

  • Apply oxidative stressors (H₂O₂, paraquat, ozone)

  • Test nitrosative stress conditions (NO donors, peroxynitrite)

  • Implement abiotic stresses (drought, salinity, extreme temperatures)

  • Design time-course experiments to track nitration dynamics

• Nitration site mapping:

  • Immunoprecipitate At4g19865 using specific antibodies

  • Analyze by mass spectrometry to identify nitrated tyrosine residues, looking for the characteristic +45 Da mass shift seen in other nitrated Arabidopsis proteins

  • Compare nitration patterns across stress treatments

  • Quantify the stoichiometry of nitration at specific sites

• Functional correlation:

  • Correlate nitration levels with At4g19865 protein activity or stability

  • Test effects of nitration on protein-protein interactions

  • Investigate subcellular localization changes upon nitration

This multi-faceted approach can reveal how nitration affects At4g19865 function and whether this modification serves as a regulatory mechanism during stress responses.

What methods can be used to analyze At4g19865 protein degradation pathways using antibodies?

As an F-box protein, At4g19865 is likely involved in protein degradation pathways and may itself be regulated by degradation. Antibody-based methods to study these processes include:

• Protein stability assessment:

  • Cycloheximide chase assays with immunoblotting to measure protein half-life

  • Pulse-chase experiments with immunoprecipitation to track synthesis and degradation rates

  • Comparative stability analysis across developmental stages or stress conditions

• Ubiquitination detection:

  • Immunoprecipitate At4g19865 and probe for ubiquitin

  • Co-immunoprecipitation with antibodies against SCF complex components

  • In vitro ubiquitination assays with recombinant proteins and immunodetection

• Proteasome inhibitor studies:

  • Compare At4g19865 levels with/without proteasome inhibitors (MG132, bortezomib)

  • Assess accumulation of ubiquitinated forms using antibody detection

  • Track subcellular redistribution upon proteasome inhibition

• SCF complex interaction analysis:

  • Co-immunoprecipitation with antibodies against Skp1, Cullin1, and Rbx1

  • Proximity ligation assays to visualize interactions in situ

  • Size exclusion chromatography followed by immunoblotting to detect complex formation

These methods provide comprehensive insights into both the role of At4g19865 in protein degradation pathways and how its own levels are regulated, which is particularly relevant given its identification in post-translational modification studies .

How can antibodies help determine the subcellular localization of At4g19865?

Determining the subcellular localization of At4g19865 using antibodies requires specialized immunolocalization approaches:

• Immunofluorescence microscopy:

  • Fix and permeabilize plant cells or tissue sections

  • Block with appropriate sera to reduce non-specific binding

  • Incubate with anti-At4g19865 primary antibodies

  • Detect with fluorescently labeled secondary antibodies

  • Counterstain with organelle markers (nuclear dyes, mitochondrial stains)

  • Image using confocal microscopy for high-resolution localization

• Subcellular fractionation with immunoblotting:

  • Isolate different cellular components (nuclei, cytosol, membranes, chloroplasts)

  • Prepare protein extracts from each fraction

  • Perform western blotting with anti-At4g19865 antibodies

  • Include markers for different compartments to validate fractionation

  • Quantify relative distribution across compartments

• Immuno-electron microscopy:

  • Prepare ultrathin sections of fixed plant tissue

  • Incubate with anti-At4g19865 antibodies

  • Detect with gold-conjugated secondary antibodies

  • Image using transmission electron microscopy

  • Quantify gold particle distribution across cellular structures

• Proximity-based approaches:

  • Use antibodies in proximity ligation assays with known organelle markers

  • Correlate localization with functional studies

  • Track changes in localization during development or stress responses

These complementary approaches provide comprehensive information about At4g19865 subcellular distribution, informing hypotheses about its function in protein degradation pathways and potential roles in stress responses related to its nitration .