At1g19160 Antibody

What is At1g19160 and why is it studied in plant research?

At1g19160 is a protein-coding gene in Arabidopsis thaliana (thale cress) that encodes an F-box family protein . F-box proteins are involved in protein-protein interactions and often function as part of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex, which targets proteins for degradation via the ubiquitin-proteasome pathway. This gene is studied for its potential roles in plant development, stress responses, and cellular signaling pathways. Understanding At1g19160 can provide insights into fundamental plant biological processes and potentially inform agricultural applications.

What applications can the At1g19160 antibody be used for?

The At1g19160 antibody has been validated for several research applications including:

The antibody can be used to study protein expression patterns, protein-protein interactions, and subcellular localization of At1g19160 in plant tissues .

What are the optimal conditions for using At1g19160 antibody in Western blot experiments?

For optimal Western blot results with At1g19160 antibody:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing 20 mM Tris pH 7.5, 5 mM MgCl₂, 2.5 mM DTT, 300 mM NaCl, 0.1% NP-40, and 1% proteasome inhibitor MG132 .

• Separation: Use 6-10% SDS-PAGE gels for optimal separation.

• Transfer: Transfer to nitrocellulose membrane for 1 hour at constant voltage.

• Blocking: Block membranes with 5% low-fat milk powder in TBS-TT (0.25% TWEEN20; 0.1% Triton-X) for 1 hour at room temperature .

• Primary antibody: Dilute At1g19160 antibody 1:5000-1:10000 in blocking buffer and incubate for 1 hour at room temperature or overnight at 4°C .

• Secondary antibody: Use anti-rabbit IgG HRP-conjugated antibody at 1:10000 dilution for 1-2 hours at room temperature .

• Detection: Use an appropriate chemiluminescence system.

The expected molecular weight is approximately 35-40 kDa, though this may vary depending on post-translational modifications.

How can I optimize immunoprecipitation experiments using At1g19160 antibody?

For successful immunoprecipitation of At1g19160:

• Prepare plant lysate in a non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA) supplemented with protease inhibitors.

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

• Incubate 2-5 μg of At1g19160 antibody with pre-cleared lysate overnight at 4°C with gentle rotation.

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

• Wash beads 4-5 times with IP wash buffer (same as lysis buffer but with 0.1% NP-40).

• Elute bound proteins by boiling in SDS sample buffer.

• Analyze by Western blot using the same or a different antibody against At1g19160, or antibodies against suspected interaction partners.

Co-immunoprecipitation can be particularly useful for studying the protein interactions of At1g19160 with other SCF complex components or potential substrates.

How can At1g19160 antibody be applied in studies of plant stress responses?

At1g19160 antibody can be a valuable tool for studying plant stress responses:

• Expression level monitoring: Using Western blot analysis to track changes in At1g19160 protein levels under various stress conditions (drought, salt, pathogen infection, etc.) .

• Protein complex dynamics: Through co-immunoprecipitation experiments, researchers can investigate how stress affects the interaction of At1g19160 with other proteins, particularly in ubiquitination pathways .

• Subcellular localization: Immunofluorescence microscopy using At1g19160 antibody can reveal changes in protein localization in response to stress .

• Chromatin immunoprecipitation (ChIP): If At1g19160 has DNA-binding capabilities or interacts with transcription factors, ChIP experiments can identify target genes regulated during stress responses, similar to approaches used for other Arabidopsis proteins .

• Immunohistochemistry: Tissue-specific expression analysis can reveal spatial patterns of At1g19160 expression in different plant organs under stress conditions.

By correlating At1g19160 expression and interactions with physiological responses, researchers can elucidate its role in stress adaptation mechanisms.

What approaches can be used to study At1g19160 protein interactions using antibody-based techniques?

Several sophisticated approaches can characterize At1g19160 protein interactions:

• Proximity-dependent biotin identification (BioID): Fuse BirA* biotin ligase to At1g19160, express in plants, and use the antibody to confirm expression before streptavidin pulldown of biotinylated proximal proteins.

• Cross-linking immunoprecipitation (CLIP): To identify RNA targets if At1g19160 binds RNA, similar to approaches used for AGO1 protein in Arabidopsis .

• Quantitative co-immunoprecipitation: Use At1g19160 antibody for pulldown followed by mass spectrometry to identify and quantify interaction partners under different conditions.

• Förster resonance energy transfer (FRET): Use fluorophore-conjugated At1g19160 antibody in combination with antibodies against suspected interaction partners for in situ detection of protein-protein interactions.

• Protein complex immunoprecipitation: Target intact protein complexes containing At1g19160 using a mild lysis buffer (e.g., 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) to preserve complex integrity.

These methods can reveal both constitutive and conditional interactions of At1g19160, providing insights into its biological functions.

How can I resolve non-specific binding issues when using At1g19160 antibody?

To minimize non-specific binding with At1g19160 antibody:

• Antibody validation: Confirm antibody specificity using positive controls (recombinant At1g19160 protein) and negative controls (knockout/knockdown plant lines) .

• Blocking optimization: Test different blocking agents beyond standard milk or BSA, such as plant-specific blocking reagents that address plant-specific background.

• Cross-adsorption: Pre-incubate the antibody with plant extract from At1g19160 knockout plants to remove antibodies that bind to non-specific epitopes.

• Titrate antibody concentration: Perform a dilution series (1:1000 to 1:20000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Increase washing stringency: Use higher salt concentrations (up to 500 mM NaCl) or add 0.1-0.2% SDS to washing buffers to reduce non-specific interactions.

• Use purified IgG fraction: If using whole antiserum, purify the IgG fraction using Protein A/G to reduce non-specific binding from other serum components.

• Epitope competition: Include excess immunizing peptide as a competitive control to confirm signal specificity.

These approaches can significantly improve signal-to-noise ratio in experiments using At1g19160 antibody.

What are the major challenges in reproducing immunolocalization results with At1g19160 antibody and how can they be addressed?

Reproducibility challenges in At1g19160 immunolocalization include:

• Fixation artifacts: Different fixatives (paraformaldehyde, glutaraldehyde) can affect epitope accessibility. Solution: Optimize fixation conditions by testing different fixatives and incubation times.

• Epitope masking: Cellular context may hide the antibody binding site. Solution: Test different antigen retrieval methods (heat-induced, enzymatic, pH-based).

• Antibody batch variation: Different lots may have varying specificities. Solution: Validate each new lot against previous ones and standardize positive controls.

• Technical variability in tissue preparation: Solution: Develop a standardized protocol for tissue embedding, sectioning, and handling.

• Plant developmental stage differences: At1g19160 expression may vary by developmental stage. Solution: Carefully document and standardize plant age and growth conditions.

• Autofluorescence interference: Plant tissues often have high autofluorescence. Solution: Use appropriate filters and background subtraction methods or consider using non-fluorescent detection methods.

• Protocol validation: Solution: Include known localization markers (e.g., nuclear, ER, or cytosolic proteins) as internal controls in each experiment.

Careful documentation of all experimental parameters is crucial for reproducible immunolocalization studies.

How does the performance of polyclonal versus monoclonal antibodies against At1g19160 compare in different experimental contexts?

For At1g19160 research, polyclonal antibodies may be preferable for initial characterization studies, while monoclonal antibodies would be advantageous for targeted domain studies or when extremely high specificity is required .

How can At1g19160 antibody studies be integrated with other omics approaches in plant research?

Integration of At1g19160 antibody data with multi-omics approaches:

• Proteomics integration:

  • Use At1g19160 antibody for immunoprecipitation followed by mass spectrometry

  • Compare antibody-based quantification with label-free quantitative proteomics

  • Validate post-translational modifications identified in proteomics studies

• Transcriptomics correlation:

  • Correlate protein levels detected by At1g19160 antibody with mRNA expression data

  • Investigate discrepancies between transcript and protein levels to identify post-transcriptional regulation

• Metabolomics connection:

  • Link At1g19160 protein levels to metabolic changes in the same tissues

  • Design experiments where At1g19160 function is perturbed and monitor both protein levels and metabolite profiles

• Phenomics integration:

  • Correlate At1g19160 protein expression patterns with phenotypic data

  • Use antibody to track protein levels in mutant lines with altered phenotypes

• Interactomics validation:

  • Validate computational predictions of protein-protein interactions using co-immunoprecipitation

  • Confirm structural models of At1g19160 interactions through antibody epitope mapping

This multi-omics integration provides a comprehensive understanding of At1g19160's role in plant biology, similar to approaches used for other plant proteins .

What are the best approaches for generating and validating novel antibodies against specific domains of At1g19160?

For developing domain-specific At1g19160 antibodies:

• Epitope selection:

  • Perform bioinformatic analysis to identify unique, accessible regions specific to At1g19160

  • Focus on domains with predicted functional significance (e.g., F-box domain, substrate-binding regions)

  • Avoid regions with high homology to other F-box proteins

• Peptide design:

  • Select 15-20 amino acid peptides with good predicted antigenicity

  • Consider adding a C-terminal cysteine for conjugation if not naturally present

  • Synthesize peptides with >85% purity for immunization

• Immunization strategy:

  • Use multiple rabbits to increase chances of success

  • Implement a long immunization schedule (12-16 weeks) for optimal response

  • Monitor antibody titers using ELISA throughout immunization

• Purification options:

  • Perform dual purification: affinity purification against the immunizing peptide followed by negative selection against similar domains from other F-box proteins

  • Alternatively, use recombinant protein domains for more native epitope presentation

• Validation requirements:

  • Test antibody recognition of recombinant full-length and domain fragments of At1g19160

  • Verify specificity using At1g19160 knockout/knockdown lines

  • Perform cross-reactivity testing against closely related F-box proteins

  • Validate in multiple applications (WB, IP, IF) with appropriate controls

This systematic approach maximizes the likelihood of generating highly specific antibodies for studying individual domains of At1g19160.

How can plant-based expression systems be optimized for the production of antibodies against At1g19160?

Optimizing plant-based antibody production against At1g19160:

• Expression system selection:

  • Arabidopsis: Provides proper post-translational modifications but moderate yield

  • Nicotiana benthamiana: Higher transient expression for rapid production

  • Glycoengineered plants: Modified to produce antibodies with humanized glycosylation

• Vector optimization:

  • Use strong promoters (e.g., CaMV 35S, ubiquitin)

  • Include appropriate targeting signals (e.g., ER retention signal KDEL) to increase accumulation

  • Optimize codon usage for the expression host

• Expression enhancement strategies:

  • Co-express with gene silencing suppressors (e.g., p19, HcPro)

  • Optimize growth conditions (temperature, light, nutrients)

  • Use molecular farming techniques for higher yields

• Purification process:

  • Implement protein A/G affinity chromatography

  • Consider adding purification tags (His, FLAG) if not affecting functionality

  • Develop plant-specific extraction buffers to minimize interference from plant compounds

• Quality control measures:

  • Assess binding affinity compared to mammalian-produced antibodies

  • Verify specificity against recombinant At1g19160

  • Test functionality in multiple assay types

Research shows that plant-expressed antibodies can achieve binding affinities and specificities comparable to mammalian-derived antibodies while offering advantages in production cost and scalability .

How might novel antibody engineering approaches enhance the utility of At1g19160 antibodies for advanced plant research?

Emerging antibody engineering approaches for At1g19160 research:

• Single-domain antibodies (nanobodies):

  • Develop smaller (~15 kDa) camelid-derived single-domain antibodies against At1g19160

  • Advantages: Better tissue penetration, stability, and access to cryptic epitopes

  • Applications: Live-cell imaging of At1g19160 dynamics, intrabody expression

• Bispecific antibodies:

  • Create antibodies that simultaneously bind At1g19160 and interacting partners

  • Applications: Detection of specific protein complexes in situ, study of conditional interactions

• Photo-crosslinking antibodies:

  • Incorporate photo-activatable amino acids into anti-At1g19160 antibodies

  • Applications: Capture transient protein interactions upon light activation

• Split-antibody complementation:

  • Develop antibody fragments that reassemble upon binding to At1g19160

  • Applications: Monitoring protein conformational changes or localization patterns

• Computationally designed antibodies:

  • Apply machine learning approaches to optimize antibody binding to specific At1g19160 epitopes

  • Applications: Higher specificity, reduced cross-reactivity with related F-box proteins

These approaches could revolutionize the study of At1g19160's dynamic functions in plant development and stress responses.

What potential roles might At1g19160 play in plant immunity, and how could antibody-based techniques help elucidate these functions?

Investigating At1g19160's role in plant immunity with antibody techniques:

• Expression pattern analysis:

  • Use At1g19160 antibody to track protein levels following pathogen exposure

  • Compare expression patterns in resistant versus susceptible plant varieties

  • Examine tissue-specific expression during immune responses

• Protein interaction studies:

  • Perform co-immunoprecipitation to identify immunity-related interaction partners

  • Look for interactions with known immune receptors or signaling components

  • Analyze how these interactions change during infection

• Post-translational modification profiling:

  • Use phospho-specific antibodies to detect activation-related modifications

  • Track ubiquitination status during immune responses (especially relevant for F-box proteins)

  • Examine how modifications affect protein localization and function

• Functional inhibition studies:

  • Develop function-blocking antibodies targeting specific domains

  • Introduce these antibodies into plant cells to disrupt At1g19160 function

  • Evaluate the impact on immune responses

• Comparative studies with immune-related F-box proteins:

  • Use antibodies against multiple F-box proteins to compare expression patterns

  • Examine co-expression and co-localization during pathogen attack