At5g56390 Antibody

What is At5g56390 and what cellular functions does it serve in Arabidopsis thaliana?

At5g56390 is an F-box/RNI-like/FBD-like domains-containing protein found in Arabidopsis thaliana. According to subcellular localization data from SUBA5, this protein is predominantly localized to the plasma membrane with a SUBAcon score of 0.784 . The protein contains 428 amino acids with a calculated molecular weight of 48,559.30 Da and an isoelectric point of 7.44 .

The protein contains several conserved domains including:

• FBD domain (InterPro:IPR013596)

• F-box domain, Skp2-like (InterPro:IPR022364)

• FBD-like domain (InterPro:IPR006566)

• Leucine-rich repeat 2 (InterPro:IPR013101)

F-box proteins typically function as components of SCF ubiquitin-ligase complexes, where they serve as substrate recognition modules. While the specific function of At5g56390 has not been fully characterized, its domain architecture suggests involvement in protein-protein interactions and potentially in protein degradation pathways.

What are the key specifications of commercially available At5g56390 antibodies?

Currently available At5g56390 antibodies are predominantly rabbit polyclonal antibodies raised against recombinant Arabidopsis thaliana At5g56390 protein. Based on product information from commercial suppliers, these antibodies have the following specifications:

These antibodies are typically available through specialized plant research antibody suppliers and are generally produced as made-to-order reagents with lead times of approximately 14-16 weeks .

What are the optimal conditions for using At5g56390 antibody in Western blot applications?

For optimal Western blot results with At5g56390 antibody, follow this methodological approach:

Sample Preparation:

• Extract total protein from Arabidopsis tissue using a specialized plant protein extraction buffer optimized for membrane proteins (as At5g56390 is primarily membrane-localized) .

• Use a buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Quantify protein concentration using Bradford assay to ensure equal loading .

Electrophoresis and Transfer:

• Load 20-30 μg of total protein per lane on a 10% SDS-PAGE gel.

• Transfer proteins to a PVDF membrane at 100V for 60 minutes in standard transfer buffer (25mM Tris, 192mM glycine, 20% methanol) .

Immunodetection:

• Block membrane in 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Incubate with primary At5g56390 antibody at 1:1000 dilution in blocking buffer overnight at 4°C.

• Wash 3 times with TBST for 10 minutes each.

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Wash 3 times with TBST for 10 minutes each.

• Develop using chemiluminescent substrate and image.

The expected band size is approximately 48-49 kDa. Including a wild-type control alongside knockout or knockdown lines is essential for validating signal specificity, similar to validation approaches used for other plant antibodies .

How can I optimize immunolocalization studies using At5g56390 antibody in plant tissues?

For successful immunolocalization of At5g56390 in plant tissues, consider this detailed protocol:

Tissue Fixation and Embedding:

• Fix fresh Arabidopsis tissue samples in 4% paraformaldehyde in PBS (pH 7.4) for 3-4 hours at 4°C.

• Wash samples 3 times in PBS (15 minutes each).

• Dehydrate through an ethanol series (30%, 50%, 70%, 90%, 100%, 100%) for 30 minutes each.

• Infiltrate with and embed in LR White resin.

• Cut 1-2 μm sections using an ultramicrotome and mount on poly-L-lysine coated slides.

Immunostaining:

• Block sections with 3% BSA in PBS for 1 hour at room temperature.

• Incubate with affinity-purified At5g56390 antibody (1:50 to 1:200 dilution) overnight at 4°C.

• Wash 3 times with PBS containing 0.1% Tween-20.

• Incubate with fluorescence-conjugated secondary antibody (e.g., Alexa Fluor 488 anti-rabbit) at 1:1000 dilution for 2 hours at room temperature.

• Wash 3 times with PBS containing 0.1% Tween-20.

• Counterstain with DAPI (1 μg/ml) for nuclear visualization.

• Mount in anti-fade mounting medium.

Controls and Validation:

• Include a negative control using pre-immune serum at the same dilution as the primary antibody.

• Use an antibody-specific blocking peptide to confirm signal specificity.

• Compare localization patterns in wild-type versus knockout/knockdown lines if available.

Research with other Arabidopsis antibodies shows that affinity purification dramatically improves the detection rate and signal specificity in immunolocalization studies .

What strategies should be employed to validate the specificity of At5g56390 antibody?

Validating antibody specificity is critical for reliable research findings. For At5g56390 antibody, implement these comprehensive validation strategies:

Genetic Validation:

• Compare Western blot or immunolocalization results between wild-type plants and At5g56390 knock-out/knock-down mutants. The specific signal should be absent or reduced in mutant lines .

• Use CRISPR-Cas9 edited lines with specific At5g56390 deletions as negative controls.

Biochemical Validation:

• Perform antigen competition assays where the antibody is pre-incubated with excess purified recombinant At5g56390 protein before application in Western blot or immunolocalization. This should abolish specific signals .

• Conduct immunoprecipitation followed by mass spectrometry to confirm that At5g56390 is the predominant protein pulled down .

Expression Pattern Validation:

• Compare protein detection patterns with known mRNA expression data from publicly available databases.

• Verify subcellular localization against predicted localization data (plasma membrane for At5g56390) .

Cross-Reactivity Assessment:

• Test the antibody against recombinant proteins with similar domains (other F-box proteins) to assess potential cross-reactivity.

• Perform Western blots on tissues from related plant species to determine antibody cross-reactivity and conservation of epitopes.

In a study of Arabidopsis antibodies, only 55% of protein antibodies could detect a signal with high confidence after rigorous validation, highlighting the importance of thorough specificity testing .

How can I troubleshoot non-specific binding issues with At5g56390 antibody?

When encountering non-specific binding with At5g56390 antibody, implement these systematic troubleshooting steps:

Antibody Purification:

• Perform antigen-affinity purification of the antibody to improve specificity. Research has shown that affinity purification "massively improved the detection rate" of plant antibodies .

• Use a column with immobilized recombinant At5g56390 protein to isolate only target-specific antibodies from the polyclonal mixture.

Blocking Optimization:

• Test different blocking agents: Compare 5% BSA, 5% non-fat dry milk, and commercial blocking buffers to identify optimal conditions.

• Extend blocking time to 2 hours at room temperature or overnight at 4°C.

Antibody Dilution Series:

• Perform a dilution series (1:500, 1:1000, 1:2000, 1:5000) to identify the optimal concentration that maximizes specific signal while minimizing background.

Buffer Modifications:

• Add 0.1-0.3% Triton X-100 to washing buffer to reduce hydrophobic interactions.

• Include 0.1-0.5M NaCl in antibody dilution buffer to diminish ionic interactions contributing to non-specific binding.

Epitope Competition:

• Pre-incubate the antibody with a peptide derived from a non-conserved region of At5g56390 prior to application.

• Perform parallel experiments with pre-immune serum to distinguish between specific and non-specific signals.

Research on antibody specificity has shown that even well-established antibodies can exhibit unexpected cross-reactivity with unrelated proteins, emphasizing the need for rigorous controls .

How can At5g56390 antibody be utilized in protein-protein interaction studies to investigate F-box protein complexes?

Investigating protein-protein interactions involving At5g56390 requires specialized approaches leveraging the antibody's properties:

Co-Immunoprecipitation (Co-IP):

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

• Incubate protein extract with At5g56390 antibody (5-10 μg) overnight at 4°C.

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

• Wash beads 4-5 times with IP buffer containing 0.1% NP-40.

• Elute bound proteins and analyze by SDS-PAGE followed by Western blotting or mass spectrometry.

• Confirm interactions by reciprocal Co-IP using antibodies against potential interacting partners.

Proximity Ligation Assay (PLA):

• Fix and permeabilize plant tissues as described for immunolocalization.

• Incubate with At5g56390 antibody and antibody against a suspected interaction partner.

• Apply PLA probes and follow manufacturer's protocol for ligation and amplification.

• Visualize interaction signals using confocal microscopy.

Pull-down Validation:

• Express recombinant At5g56390 with a tag (His, GST) in E. coli.

• Immobilize on appropriate resin and incubate with plant extracts.

• Wash and elute bound proteins.

• Confirm interactions using At5g56390 antibody and antibodies against putative partners.

This approach has been successfully employed to characterize protein-protein interactions in other plant systems and could reveal components of SCF complexes containing At5g56390 .

What considerations are important when using At5g56390 antibody for chromatin immunoprecipitation (ChIP) experiments?

Although At5g56390 is not directly documented as a DNA-binding protein, if investigating potential chromatin associations of this F-box protein, consider these methodological adaptations:

Chromatin Preparation:

• Cross-link plant material with 1% formaldehyde for 10 minutes under vacuum.

• Quench with 0.125M glycine for 5 minutes.

• Extract nuclei using a plant-specific nuclear isolation buffer containing 0.25M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl₂, 1% Triton X-100, and protease inhibitors.

• Sonicate chromatin to obtain fragments of 200-500 bp.

Immunoprecipitation:

• Pre-clear chromatin with protein A beads and non-immune IgG.

• Incubate chromatin with 5-10 μg of affinity-purified At5g56390 antibody overnight at 4°C.

• Add protein A beads and incubate for 3 hours.

• Perform stringent washes with increasing salt concentrations.

• Reverse cross-links and purify DNA.

Controls and Validation:

• Include IgG control to establish background signal.

• Perform parallel ChIP with an established chromatin-associated protein antibody as positive control.

• Include input sample (non-immunoprecipitated chromatin) in all analyses.

• Validate enrichment of specific genomic regions by qPCR before proceeding to ChIP-seq.

Data Analysis:

• For ChIP-seq data, use appropriate peak-calling algorithms optimized for plant genomes.

• Compare binding patterns with known transcription factors regulating related biological processes.

• Validate selected targets using ChIP-qPCR.

ChIP applications would be considered advanced and exploratory for At5g56390, requiring careful optimization and validation .

What are the critical factors to consider when designing new epitope-specific antibodies against At5g56390 for particular domains?

When designing domain-specific antibodies for At5g56390, consider these critical factors:

Epitope Selection Strategy:

• Perform comprehensive sequence analysis to identify unique regions within specific domains (F-box, FBD, or LRR domains).

• Avoid regions with high sequence similarity to other F-box family proteins (>40% similarity) .

• Select epitopes with favorable biophysical properties:

  • Hydrophilicity score >0.5

  • Surface accessibility >50%

  • Flexibility index >0.5

  • Low tendency to form secondary structures

Comparison of Peptide vs. Recombinant Protein Approaches:

• For domain-specific antibodies, recombinant protein approaches show significantly higher success rates (55%) compared to peptide approaches (<5%) .

• When using the recombinant protein approach:

  • Express individual domains separately

  • Verify proper folding using circular dichroism

  • Confirm purity >90% by SDS-PAGE

Post-Production Evaluation:

• Perform dot blot titration against the immunogen to confirm antibody titer and sensitivity (detection in picogram range) .

• Assess cross-reactivity against related domains from other F-box proteins.

• Validate domain specificity using truncated protein constructs expressing different domains of At5g56390.

Research on Arabidopsis antibodies shows that careful epitope selection and rigorous validation are critical for success, as only about 55% of antibodies raised against recombinant proteins could detect signals with high confidence .

How can computational approaches be integrated with At5g56390 antibody studies to improve antibody design and validation?

Integrating computational approaches with experimental antibody research offers powerful advantages:

Computational Epitope Prediction:

• Implement machine learning algorithms trained on known plant protein epitopes to predict optimal antigenic regions of At5g56390.

• Use protein 3D structure prediction tools (AlphaFold) to identify surface-exposed regions likely to generate accessible epitopes.

• Apply B-cell epitope prediction algorithms that consider:

  • Surface accessibility

  • Hydrophilicity

  • Flexibility

  • Secondary structure propensity

Sequence Conservation Analysis:

• Perform multiple sequence alignments of F-box proteins across plant species to identify:

  • Highly conserved regions (for cross-species reactivity)

  • Unique regions (for At5g56390 specificity)

• Calculate conservation scores for each residue using the ConSurf server to guide epitope selection.

Antibody-Antigen Interaction Modeling:

• Use molecular docking simulations to predict antibody-antigen binding interactions.

• Simulate binding energy landscapes to identify optimal paratope-epitope pairs.

• Apply in silico affinity maturation techniques similar to those used in therapeutic antibody development .

Integration with Experimental Data:

• Create feedback loops between computational predictions and experimental validation data.

• Develop a database of successful and unsuccessful epitopes for plant antibodies to refine prediction algorithms.

• Incorporate epitope mapping data to validate computational models.

Recent computational antibody design approaches have successfully restored and enhanced antibody potency against variant targets, demonstrating the power of integrating simulation and machine learning in antibody engineering .

What quality control testing should be performed on At5g56390 antibody lots to ensure reproducibility across experiments?

To ensure consistent antibody performance across experiments, implement this comprehensive quality control testing strategy:

Initial Characterization:

• Determine protein concentration using BCA or Bradford assay.

• Assess purity by SDS-PAGE (>90% pure in the IgG band).

• Verify immunoglobulin integrity using capillary electrophoresis.

Functional Testing:

• Perform dot blot titration against recombinant At5g56390 protein to determine:

  • Detection limit (should detect in picogram range)

  • EC50 value (concentration required for 50% maximal signal)

• Conduct Western blot against plant extract containing At5g56390:

  • Confirm correct band size (approximately 48.5 kDa)

  • Evaluate signal-to-noise ratio (>5:1 is acceptable)

• Test lot-to-lot consistency by comparing with reference standard:

  • Titer should be within ±25% of reference

  • Specificity pattern should be consistent

Documentation and Standardization:

According to FDA guidance on immunogenicity testing, proper characterization of antibody reagents is critical for ensuring reliable and reproducible results .

How should researchers document and report At5g56390 antibody validation to ensure scientific reproducibility?

Proper documentation and reporting of antibody validation is crucial for scientific reproducibility. Follow these best practices:

Essential Documentation Elements:

• Complete antibody identification information:

  • Vendor and catalog number

  • Lot number

  • Host species and clonality

  • Immunogen details (full sequence if available)

  • Production and purification methods

• Validation experiments performed:

  • Western blot images showing full blots with molecular weight markers

  • Validation in knockout/knockdown lines

  • Peptide competition assays

  • Cross-reactivity tests

Standardized Reporting Format:

• Include a dedicated "Antibody Validation" section in the Methods that contains:

  • All antibodies used and their sources

  • Detailed validation methodology

  • Controls included

  • Exact dilutions and incubation conditions

• Provide access to raw validation data via data repositories or supplementary materials.

Application-Specific Validation:

• For each distinct application (Western blot, immunolocalization, etc.), document:

  • Specific validation performed for that application

  • Optimization steps undertaken

  • Known limitations or caveats

Adherence to Reporting Guidelines:

• Follow the "Minimum Information About a Protein Affinity Reagent" (MIAPAR) guidelines.

• Implement recommendations from the International Working Group for Antibody Validation.

• Cite previous validations if building upon established antibody characterization.

Research has demonstrated that antibody reagents require comprehensive validation and documentation to ensure reproducibility, particularly in plant systems where validation resources are more limited than in mammalian research .

How might emerging single-cell technologies be combined with At5g56390 antibody for advanced spatial proteomics in plant tissues?

Integrating At5g56390 antibody with cutting-edge single-cell technologies offers novel insights into protein distribution and dynamics:

Single-Cell Imaging Mass Cytometry:

• Conjugate At5g56390 antibody with rare earth metals (e.g., lanthanides).

• Prepare thin plant tissue sections (5-8 μm) on specialized slides.

• Stain with metal-conjugated antibody panel including At5g56390 and other markers.

• Analyze using a CyTOF-based imaging system to achieve subcellular resolution.

• Quantify protein expression at single-cell level while preserving spatial context.

Multiplexed Immunofluorescence:

• Employ cyclic immunofluorescence with At5g56390 antibody:

  • Stain tissue with At5g56390 antibody and fluorescent secondary

  • Image the tissue

  • Chemically inactivate fluorophores

  • Repeat with different antibodies (10-50 cycles)

• Reconstruct protein interaction networks at subcellular resolution.

• Correlate At5g56390 localization with other F-box proteins and SCF complex components.

In situ Proximity Ligation:

• Apply At5g56390 antibody in combination with antibodies against putative interaction partners.

• Analyze spatial distribution of protein-protein interactions at single-cell resolution.

• Correlate interaction patterns with cell types and developmental stages.

Single-Cell Western Blotting:

• Prepare protoplasts from Arabidopsis tissues.

• Isolate single cells in microwell arrays.

• Perform in-situ cell lysis, protein separation, and immunoblotting with At5g56390 antibody.

• Quantify protein expression in individual cells.

These advanced approaches build upon established antibody methodologies but extend them to single-cell resolution, revealing cell-to-cell variation in protein expression and interactions .

What are the possibilities and limitations of using At5g56390 antibody in combination with CRISPR-Cas9 genome editing technologies?

Combining At5g56390 antibody with CRISPR-Cas9 technologies creates powerful experimental systems:

Antibody Validation Through CRISPR:

• Generate precise At5g56390 knockout lines using CRISPR-Cas9.

• Create domain-specific deletions to map antibody epitopes in vivo.

• Introduce epitope tags at the endogenous locus for parallel detection with commercial tag antibodies.

• Use these engineered lines as gold-standard controls for antibody specificity.

Functional Proteomics Applications:

• Create allelic series of At5g56390 variants with domain-specific modifications:

  • F-box domain mutations

  • FBD domain deletions

  • LRR modifications

• Use the At5g56390 antibody to assess:

  • Protein stability changes

  • Subcellular localization alterations

  • Complex formation differences

Proximity Proteomics Integration:

• Use CRISPR to fuse proximity labeling enzymes (BioID, APEX) to At5g56390.

• Apply the At5g56390 antibody to verify correct fusion protein expression.

• Identify proximal proteins in living cells to map the At5g56390 interaction landscape.

Limitations and Considerations:

• Epitope disruption: CRISPR-induced mutations near antibody epitopes may affect recognition.

• Tag interference: Adding tags may alter protein function or localization.

• Expression differences: CRISPR-modified genes may show altered expression levels requiring antibody detection optimization.

• Off-target effects: Potential CRISPR off-targets require careful validation with antibodies.

This integrated approach has been successfully applied to validate other plant antibodies and study protein function in vivo, providing a powerful framework for At5g56390 research .

How does the performance of At5g56390 antibody compare with fluorescent protein fusions for localization studies?

When choosing between antibody-based detection and fluorescent protein fusions for At5g56390 studies, consider these comparative factors:

Detection Sensitivity Comparison:

Technical Considerations:

• Native Protein Detection:

  • Antibody: Detects endogenous protein without modification

  • FP Fusion: Requires genetic modification, potential functional disruption

• Temporal Resolution:

  • Antibody: Fixed-time analysis, no real-time dynamics

  • FP Fusion: Allows live-cell imaging and protein dynamics studies

• Spatial Resolution:

  • Antibody: Limited by fixation artifacts and antibody accessibility

  • FP Fusion: Limited by diffraction but allows super-resolution approaches

• Experimental Flexibility:

  • Antibody: Requires fixation, compatible with co-localization studies

  • FP Fusion: Permits FRAP, FRET, BiFC, and other advanced imaging techniques

Evidence-Based Recommendations:

• For fixed tissues and protein abundance studies, affinity-purified At5g56390 antibody provides reliable detection of endogenous protein .

• For dynamic studies of protein movement or interaction, C-terminal FP fusions are preferred, as N-terminal fusions may disrupt the F-box domain function.

• For highest confidence, use both approaches complementarily:

  • Verify FP fusion localization patterns with antibody staining

  • Confirm antibody specificity using FP fusion as positive control

Research with other plant proteins demonstrates that complementary use of antibodies and fluorescent proteins provides the most comprehensive understanding of protein localization and function .

What are the comparative advantages of proteomics approaches versus antibody-based methods for studying At5g56390?

When investigating At5g56390, researchers should understand the complementary nature of antibody-based and proteomics approaches:

Comparative Analysis of Detection Methods:

Methodological Strengths and Limitations:

• Protein Identification:

  • Antibody: Limited to target protein and known interactions

  • Proteomics: Unbiased identification of entire complexes and networks

• Protein Modifications:

  • Antibody: Requires modification-specific antibodies

  • Proteomics: Can identify novel modifications and quantify stoichiometry

• Interaction Studies:

  • Antibody: Excellent for targeted validation of specific interactions

  • Proteomics: Superior for discovering novel interaction partners

• Subcellular Localization:

  • Antibody: Excellent spatial resolution in tissues

  • Proteomics: Better for global proteome mapping of isolated organelles

Integrated Research Strategy:

• Use proteomics for discovery phase:

  • Identify At5g56390 expression patterns across tissues/conditions

  • Discover potential interaction partners

  • Map post-translational modifications

• Use At5g56390 antibody for validation and detailed characterization:

  • Confirm protein localization in specific cell types

  • Validate interactions identified by proteomics

  • Study protein dynamics in specific experimental conditions