At1g47702 Antibody

What is At1g47702 and why is it significant in plant research?

At1g47702 is a gene locus in Arabidopsis thaliana that encodes a putative F-box protein . F-box proteins are significant components of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex, which plays crucial roles in protein degradation and various signaling pathways in plants. This particular F-box protein is of interest to researchers studying protein-protein interactions, developmental processes, and stress responses in Arabidopsis and related species.

The antibody against At1g47702 protein allows for the detection, quantification, and localization of this protein in plant tissues, making it an essential tool for investigating protein function, expression patterns, and regulatory mechanisms in plant molecular biology and genetics research.

How is the At1g47702 antibody typically generated?

At1g47702 antibodies are typically generated through a polyclonal approach using rabbits as hosts . The process involves:

• Selecting appropriate immunogenic peptides derived from the At1g47702 protein sequence

• Conjugating these peptides to carrier proteins like KLH (Keyhole Limpet Hemocyanin)

• Immunizing rabbits with the conjugated peptide

• Collecting serum after sufficient antibody production

• Purifying the antibodies through affinity chromatography or other methods

• Validating specificity through Western blotting and other techniques

This approach is similar to the generation of other plant protein antibodies, such as those described for FtsH protein variants in Arabidopsis thaliana .

What are the primary differences between polyclonal and monoclonal antibodies for plant protein research?

For At1g47702 research, polyclonal antibodies are typically preferred due to their ability to recognize multiple epitopes, which can be advantageous when working with plant tissues where protein conformation or post-translational modifications may affect epitope accessibility .

What are the optimal conditions for using At1g47702 antibody in Western blotting of plant samples?

Based on similar plant protein antibody protocols , the following conditions are recommended for Western blotting with At1g47702 antibody:

• Sample preparation:

  • Extract total protein from plant tissue using a buffer containing 0.2M Tris-HCl pH 6.8, 2% SDS, 10% mercaptoethanol, and 5M urea

  • Typical loading: 8-10 μg of total protein per lane

• Gel electrophoresis:

  • 12% SDS-PAGE is typically sufficient for resolving At1g47702 protein

  • Include molecular weight markers appropriate for the expected size range

• Transfer conditions:

  • Transfer to nitrocellulose or PVDF membrane at 100V for 1-2 hours or 25V overnight

  • Confirm transfer efficiency with reversible staining

• Blocking:

  • Block with 5% non-fat milk in PBS-T or TBS-T for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute At1g47702 antibody 1:500 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing and secondary antibody:

  • Wash 3 × 5-10 minutes with PBS-T or TBS-T

  • Incubate with HRP-conjugated anti-rabbit IgG (1:5000-1:10000) for 1-2 hours at room temperature

• Detection:

  • Develop using ECL substrate

  • Typical exposure time: 1-3 minutes, depending on expression level

These conditions should be optimized based on the specific properties of your plant material and antibody lot .

How can I perform immunolocalization of At1g47702 protein in plant tissues?

For immunolocalization of At1g47702 in plant tissues, the following methodology is recommended:

• Tissue fixation:

  • Fix fresh plant tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours

  • Alternative: Use 3:1 ethanol:acetic acid for better preservation of cellular morphology

• Embedding and sectioning:

  • Dehydrate tissues through an ethanol series

  • Embed in paraffin or suitable resin

  • Section at 5-10 μm thickness onto adhesive slides

• Antigen retrieval:

  • Dewax sections and rehydrate

  • Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Cool slowly to room temperature

• Blocking and antibody incubation:

  • Block with 3% BSA, 5% normal goat serum in PBS for 1 hour

  • Incubate with At1g47702 antibody (1:100 to 1:500) overnight at 4°C

  • Wash 3 × 10 minutes with PBS-T

  • Incubate with fluorophore-conjugated secondary antibody (1:200 to 1:500) for 2 hours at room temperature

  • Wash 3 × 10 minutes with PBS-T

• Counterstaining and mounting:

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

  • Mount in anti-fade medium

• Controls:

  • Include negative controls (no primary antibody)

  • Use tissues from knockout or knockdown plants if available

  • Pre-absorb antibody with immunizing peptide as specificity control

This protocol can be adapted from methods used for other plant proteins and should be optimized for the specific tissues being examined.

What approaches can be used to validate the specificity of At1g47702 antibody?

Multiple approaches should be employed to validate the specificity of At1g47702 antibody:

• Western blot analysis:

  • Compare wild-type plants with At1g47702 knockout/knockdown lines

  • Observe a band of the expected molecular weight in wild-type that is reduced or absent in mutant lines

  • Test cross-reactivity with related F-box proteins

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Perform Western blot in parallel with untreated antibody

  • Specific signals should be significantly reduced or eliminated

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate proteins using At1g47702 antibody

  • Identify pulled-down proteins by mass spectrometry

  • Confirm presence of At1g47702 protein and evaluate non-specific binding

• Immunohistochemistry in transgenic lines:

  • Compare immunostaining patterns between wild-type and At1g47702-GFP fusion lines

  • Signal patterns should overlap, confirming antibody specificity

• Cross-species reactivity:

  • Test reactivity in related species with known sequence homology

  • Correlate signal intensity with sequence conservation

This multi-method validation approach ensures robust confirmation of antibody specificity before proceeding with experimental applications .

How should I interpret variations in At1g47702 protein detection across different plant tissues or conditions?

When interpreting variations in At1g47702 protein detection across different experimental conditions, consider:

• Biological factors:

  • Tissue-specific expression patterns are common for F-box proteins

  • Developmental stage can significantly impact expression levels

  • Stress conditions may induce or repress expression

  • Post-translational modifications might affect antibody recognition

• Technical considerations:

  • Normalize protein loading using appropriate housekeeping proteins

  • Consider using total protein normalization (e.g., Ponceau S staining) as an alternative

  • Validate results using RT-qPCR to correlate protein and transcript levels

  • Perform biological replicates (n ≥ 3) to establish statistical significance

• Quantification approach:

  • Use densitometry software for Western blot quantification

  • Establish a standard curve using recombinant protein if absolute quantification is needed

  • Apply appropriate statistical analyses for comparisons across conditions

Substantial variation in At1g47702 levels might indicate biological regulation worthy of further investigation, potentially through time-course experiments or analysis under different environmental stimuli .

What are common troubleshooting strategies for weak or non-specific signals when using At1g47702 antibody?

When encountering weak or non-specific signals with At1g47702 antibody, implement these troubleshooting strategies:

For particularly challenging samples, consider using enhanced chemiluminescence substrates with higher sensitivity or switching to fluorescent secondary antibodies for improved signal-to-noise ratios .

How can I distinguish between specific and non-specific binding in immunohistochemistry experiments using At1g47702 antibody?

To distinguish between specific and non-specific binding in immunohistochemistry:

• Essential controls:

  • Omit primary antibody (secondary antibody only control)

  • Use pre-immune serum at the same concentration as the antibody

  • Include tissue from At1g47702 knockout/knockdown plants

  • Perform peptide competition by pre-incubating antibody with immunizing peptide

• Signal evaluation:

  • Specific signal should be absent in knockout tissues and reduced in knockdown lines

  • Signal should be significantly reduced or eliminated in peptide competition assays

  • Signal pattern should be consistent with predicted subcellular localization

  • Evaluate signal in tissues known to express and not express the target

• Advanced validation:

  • Compare immunolocalization pattern with fluorescent protein fusion localization

  • Use dual labeling with antibodies against known interacting partners

  • Apply super-resolution microscopy techniques for precise localization assessment

• Documentation:

  • Image all samples using identical acquisition parameters

  • Present controls alongside experimental samples in publications

  • Quantify signal intensity across different samples for objective comparison

These approaches will help establish the specificity of immunohistochemical signals and provide confidence in the observed localization patterns .

How can At1g47702 antibody be used in protein-protein interaction studies?

At1g47702 antibody can be employed in several protein-protein interaction approaches:

• Co-immunoprecipitation (Co-IP):

  • Use At1g47702 antibody to pull down the protein complex from plant extracts

  • Identify interacting partners by Western blot or mass spectrometry

  • Perform reciprocal Co-IP with antibodies against suspected interacting proteins

  • Include appropriate controls (IgG control, knockout plant extracts)

• Proximity-dependent biotin identification (BioID):

  • Generate fusion of At1g47702 with a promiscuous biotin ligase

  • Express the fusion protein in plants

  • Use streptavidin pull-down to isolate biotinylated proteins

  • Validate interactions using At1g47702 antibody in Western blots

• Chromatin immunoprecipitation (ChIP):

  • For potential transcription factor interactions

  • Cross-link protein-DNA complexes in plant tissues

  • Immunoprecipitate using At1g47702 antibody

  • Identify associated DNA sequences by qPCR or sequencing

• In situ proximity ligation assay (PLA):

  • Combine At1g47702 antibody with antibody against potential interacting protein

  • Use species-specific secondary antibodies with attached oligonucleotides

  • Amplify signal when proteins are in close proximity (<40 nm)

  • Visualize interaction sites in intact cells

These methods allow for the investigation of protein complexes involving At1g47702 protein under physiological conditions and can reveal novel insights into its functional role in plant cellular processes .

What are the considerations for using At1g47702 antibody in quantitative proteomics studies?

For incorporating At1g47702 antibody in quantitative proteomics studies:

• Immunoprecipitation-based proteomics:

  • Optimize immunoprecipitation conditions specifically for mass spectrometry compatibility

  • Use magnetic beads conjugated with At1g47702 antibody for cleaner preparations

  • Consider crosslinking antibody to beads to prevent antibody contamination

  • Include appropriate negative controls (non-specific IgG, knockout samples)

• Sample preparation considerations:

  • Minimize keratin contamination during sample handling

  • Perform on-bead digestion to reduce sample loss

  • Consider using FASP (Filter-Aided Sample Preparation) for cleaner peptide preparations

  • Include internal standard peptides for quantification

• Data analysis approaches:

  • Use label-free quantification or isotope labeling (SILAC, TMT) for comparative studies

  • Apply appropriate statistical methods for data normalization and analysis

  • Account for sample complexity when interpreting results

  • Validate key findings with targeted proteomics approaches (SRM/MRM)

• Validation of proteomics results:

  • Confirm key protein interactions by co-immunoprecipitation followed by Western blotting

  • Use recombinant protein standards for absolute quantification

  • Correlate protein abundance changes with transcript levels where appropriate

These considerations will help ensure robust and reproducible results when using At1g47702 antibody for proteomics applications .

How does the epitope selection affect the performance of At1g47702 antibody in different experimental contexts?

The epitope selection has profound effects on antibody performance across different applications:

• Structural considerations:

  • N-terminal epitopes may be more accessible in native proteins but could be blocked by signal peptides

  • C-terminal epitopes might be recognized in both full-length and truncated forms

  • Internal epitopes from conserved domains may cross-react with related F-box proteins

  • Epitopes from hydrophilic regions are generally more immunogenic and accessible

• Application-specific impacts:

• Experimental evidence:

  • Data from similar plant protein antibodies show that epitope selection significantly affects sensitivity and specificity

  • Antibodies raised against different epitopes of the same protein can show distinct localization patterns

  • Post-translational modifications near the epitope can dramatically affect antibody recognition

• Strategic approaches:

  • Use antibodies targeting different epitopes to validate results

  • Consider developing epitope-specific antibodies when studying protein variants

  • For comprehensive detection, combine antibodies against different epitopes

Understanding these considerations is crucial when selecting or generating At1g47702 antibodies for specific research applications .

What are the latest advanced approaches for increasing sensitivity and specificity of At1g47702 detection in plant samples?

Recent methodological advances for enhanced At1g47702 detection include:

• Signal amplification technologies:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold for immunohistochemistry

  • Polymer-based detection systems enhance signal without increasing background

  • Quantum dots as alternative to traditional fluorophores provide brighter, more stable signals

  • Proximity ligation assays for ultra-sensitive detection with reduced background

• Antibody engineering approaches:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Recombinant nanobodies derived from camelid antibodies for accessing restricted epitopes

  • Bi-specific antibodies combining At1g47702 recognition with secondary signal generators

  • Affinity maturation techniques to enhance binding properties

• Sample preparation innovations:

  • Optimized antigen retrieval protocols specific for plant tissues

  • Clearing techniques for whole-mount immunostaining of plant tissues

  • Specialized extraction buffers for membrane-associated proteins

  • Subcellular fractionation to enrich low-abundance targets

• Computational and multiplexing approaches:

  • Automated image analysis for quantitative immunohistochemistry

  • Spectral unmixing for simultaneous detection of multiple targets

  • Machine learning algorithms for signal pattern recognition

  • Correlative microscopy combining immunolocalization with structural imaging

These advanced approaches can significantly improve the detection of challenging targets like At1g47702, especially in complex plant tissues or when protein abundance is low .

How can researchers address contradictory results when comparing At1g47702 antibody data with gene expression or mutant phenotype analyses?

When faced with contradictory results between antibody-based detection and other experimental approaches:

• Systematic validation:

  • Verify antibody specificity using knockout/knockdown lines

  • Confirm protein detection using multiple antibodies targeting different epitopes

  • Use complementary methods like MS/MS to validate protein identity

  • Test for post-translational modifications that might affect antibody recognition

• Transcript-protein correlation analysis:

  • Consider time delays between transcription and protein accumulation

  • Investigate potential post-transcriptional regulation mechanisms

  • Examine protein stability and turnover rates

  • Assess translation efficiency through polysome profiling

• Technical considerations:

  • Evaluate sensitivity limitations of different detection methods

  • Consider spatial and temporal expression patterns that might differ between methods

  • Assess potential interference from closely related proteins

  • Examine technical variability across experimental replicates

• Biological interpretation framework:

  • Protein function might be regulated post-translationally without changes in abundance

  • Compensatory mechanisms may exist in mutant lines

  • Protein localization changes might occur without abundance changes

  • Consider redundancy among related F-box proteins

• Integrative approaches:

  • Combine multiple independent methods to build consensus

  • Use proteomics approaches to determine absolute protein quantities

  • Develop reporter systems to monitor protein activity rather than just abundance

  • Consider mathematical modeling to reconcile seemingly contradictory datasets

This systematic approach can help resolve apparent contradictions and lead to deeper biological insights regarding At1g47702 function .

How might At1g47702 antibodies contribute to understanding plant immune responses and disease resistance?

Recent research suggests potential roles for F-box proteins like At1g47702 in plant immunity, offering several research directions:

• Protein dynamics during pathogen challenge:

  • At1g47702 antibodies can track protein abundance changes during infection

  • Immunolocalization can reveal subcellular redistribution upon pathogen recognition

  • Co-immunoprecipitation can identify interaction partners specific to immune response

  • Phospho-specific antibodies could detect activation-related modifications

• Connections to established immune pathways:

  • F-box proteins often regulate stability of immune receptors and signaling components

  • At1g47702 may participate in proteasome-mediated degradation of negative regulators

  • Antibodies can help establish the position of At1g47702 within immune signaling cascades

  • Cross-species studies could reveal conservation of immune functions

• Experimental approaches:

  • Time-course studies following pathogen exposure or immune elicitor treatment

  • Comparative analyses between resistant and susceptible plant varieties

  • Simultaneous detection of At1g47702 and known immune markers

  • Analysis of protein-protein interactions specific to infection contexts

Understanding the role of At1g47702 in immunity could potentially contribute to developing crops with enhanced disease resistance, highlighting the broader impact of this fundamental research .

What are the methodological considerations for designing cross-species studies using At1g47702 antibody?

When extending At1g47702 antibody applications across plant species:

• Sequence analysis prerequisites:

  • Perform thorough sequence alignment of At1g47702 homologs across target species

  • Identify regions of high conservation for optimal epitope selection

  • Assess potential cross-reactivity with related proteins within each species

  • Consider developing consensus peptide-based antibodies for broader reactivity

• Validation requirements:

  • Confirm specificity in each species separately using Western blotting

  • Include appropriate positive and negative control tissues

  • Determine optimal working dilutions that may differ between species

  • Validate subcellular localization patterns in comparison to predicted localization

• Experimental design considerations:

  • Include Arabidopsis samples as reference standards

  • Adapt extraction protocols for species-specific tissues

  • Optimize incubation conditions for each species

  • Prepare species-appropriate blocking solutions to minimize background

• Data interpretation framework:

  • Consider evolutionary relationships when comparing signal intensities

  • Account for differences in protein abundance across species

  • Correlate antibody reactivity with sequence conservation at epitope

  • Interpret localization differences in context of species-specific cellular organization

Cross-species approaches can provide valuable evolutionary insights into F-box protein conservation and diversification, potentially revealing specialized functions that evolved in different plant lineages .

How can computational approaches enhance the design and application of next-generation At1g47702 antibodies?

Computational methods are revolutionizing antibody development and application:

• Epitope prediction and optimization:

  • Machine learning algorithms can predict optimal epitopes based on:

    • Surface accessibility

    • Immunogenicity

    • Specificity across related proteins

    • Stability under experimental conditions

  • Molecular dynamics simulations can model epitope behavior in different environments

  • Structural biology approaches can identify conformational epitopes

• Antibody engineering:

  • In silico affinity maturation to enhance binding properties

  • Computational design of recombinant antibody fragments with improved characteristics

  • Structure-based optimization of antibody-antigen interfaces

  • Prediction of developability properties (solubility, stability, yield)

• Cross-reactivity analysis:

  • Proteome-wide scanning for potential cross-reactive epitopes

  • Simulation of antibody binding to related F-box proteins

  • Prediction of species cross-reactivity based on sequence conservation

  • Identification of potential post-translational modification sites affecting recognition

• Data integration platforms:

  • Systems for integrating antibody validation data across multiple experiments

  • Automated analysis of immunolocalization patterns

  • Standardized reporting formats for antibody characteristics

  • Machine learning for pattern recognition in complex datasets

These computational approaches can significantly improve antibody design and application, leading to more specific and versatile tools for At1g47702 research .

What are the current best practices for reproducibility when using At1g47702 antibody in plant research?

To ensure reproducibility in At1g47702 antibody-based research:

• Comprehensive antibody reporting:

  • Document complete antibody information:

    • Source, catalog number, lot number

    • Host species and antibody type (polyclonal/monoclonal)

    • Target epitope sequence

    • Validation method references

  • Include images of full unedited blots in supplementary materials

  • Report all experimental conditions in detail

• Validation standards:

  • Perform specificity tests with appropriate controls for each new application

  • Include knockout/knockdown validation whenever possible

  • Document cross-reactivity testing with related proteins

  • Validate performance in each plant species studied

• Experimental design rigor:

  • Include all relevant controls in each experiment

  • Use biological replicates (n ≥ 3) and technical replicates

  • Blind analysis when possible, particularly for subjective assessments

  • Pre-register experimental protocols when feasible

• Data sharing:

  • Deposit raw data in appropriate repositories

  • Share detailed protocols through platforms like protocols.io

  • Report negative results to counter publication bias

  • Consider open peer review to enhance transparency