At1g31090 Antibody

How can I validate the specificity of an At1g31090 antibody?

Validating antibody specificity for At1g31090 requires a multi-step approach. Begin by examining the antibody's documented immunogen sequence and compare it to the reference protein sequence of At1g31090 (Entrez Gene ID: 839995). Ensure the antibody was raised against either the full-length protein or a fragment unique to At1g31090 rather than conserved regions shared with other F-box proteins .

For experimental validation, employ the following methods:

• Western blot analysis using plant tissue extracts from wild-type and At1g31090 knockout/knockdown plants

• Immunoprecipitation followed by mass spectrometry

• Testing against recombinant At1g31090 protein

• Pre-adsorption tests with the immunizing peptide/protein

The antibody should recognize a protein of the expected molecular weight (~32 kDa for At1g31090) and show reduced or absent signal in knockout/knockdown lines .

What controls should I include when using an At1g31090 antibody in immunoblotting experiments?

For Arabidopsis tissue samples, always include wild-type Col-0 as a reference standard and compare expression levels across different tissues as At1g31090 may be differentially expressed . When challenging results occur, consider testing multiple antibody lots or sourcing antibodies from different suppliers targeting distinct epitopes of the protein.

What are the optimal fixation and tissue preparation methods for immunolocalization of At1g31090?

For successful immunolocalization of At1g31090, tissue preparation is critical. The optimal protocol depends on the plant tissue type and developmental stage. For Arabidopsis seedlings and leaves:

• Fix tissue in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at room temperature or overnight at 4°C

• Perform permeabilization with 0.1% Triton X-100 in PBS for 15-30 minutes

• Block with 3-5% BSA in PBS for at least 1 hour

For root tissue:

• Use a shorter fixation time (1-2 hours) to preserve antigenicity

• Consider incorporating 0.1% glutaraldehyde for improved structural preservation if protein antigenicity allows

For F-box proteins like At1g31090 that may be part of protein complexes, gentler fixation protocols may be necessary to preserve epitope accessibility. Always optimize the protocol by testing different fixation times and concentrations to balance structural preservation with antibody accessibility .

How can I determine the subcellular localization of At1g31090 using antibodies?

Determining the subcellular localization of At1g31090 requires a systematic approach combining multiple techniques:

• Immunofluorescence microscopy: Use the validated At1g31090 antibody in conjunction with organelle-specific markers (nuclear, cytosolic, membrane, etc.). Perform z-stack imaging to accurately determine colocalization patterns.

• Cell fractionation followed by immunoblotting: Separate plant cell components (cytosol, nucleus, membrane fractions, etc.) through differential centrifugation and detect At1g31090 in each fraction using the validated antibody. This approach provides quantitative data about the protein's distribution.

• Immuno-electron microscopy: For high-resolution localization, use gold-conjugated secondary antibodies to visualize At1g31090 at the ultrastructural level.

As an F-box protein, At1g31090 may participate in protein degradation pathways and could show dynamic localization depending on cellular conditions. Compare results from multiple techniques and developmental stages to build a comprehensive localization profile .

What are the best methods for detecting protein-protein interactions involving At1g31090?

F-box proteins like At1g31090 typically function within SCF ubiquitin ligase complexes and interact with multiple proteins. To investigate these interactions:

When using antibody-based approaches like co-IP, ensure your At1g31090 antibody doesn't interfere with protein-protein interaction interfaces. For F-box proteins, consider using N-terminal or C-terminal epitope tags if the antibody quality is questionable, though verify these tags don't affect protein function .

How can I measure At1g31090 protein levels under different experimental conditions?

Quantifying At1g31090 protein levels across experimental conditions requires careful consideration of the protein's characteristics. As an F-box protein, At1g31090 may have relatively low expression levels and potentially short half-life due to its involvement in protein degradation pathways.

For accurate quantification:

• Quantitative Western Blotting: Use infrared fluorescence or chemiluminescence detection with a standard curve of recombinant At1g31090 protein. Normalize to loading controls appropriate for your experimental conditions.

• ELISA: Develop a sandwich ELISA using two antibodies recognizing different epitopes of At1g31090, or use a capture antibody against At1g31090 and a detection antibody against a tag if using transgenic plants.

• Selected Reaction Monitoring (SRM) Mass Spectrometry: For absolute quantification, develop an SRM assay using synthetic peptides representing unique regions of At1g31090.

When examining stress responses or developmental changes, create a time course to capture transient changes in protein expression. Compare protein levels with transcript levels to identify post-transcriptional regulation mechanisms .

Why might I observe multiple bands when probing for At1g31090 in Western blots?

Multiple bands in Western blots when probing for At1g31090 can have several causes that require systematic investigation:

• Post-translational modifications: F-box proteins often undergo modifications like phosphorylation, ubiquitination, or SUMOylation that alter their electrophoretic mobility. Use phosphatase treatment or deubiquitinating enzymes to test this hypothesis.

• Alternative splicing: Check databases for documented splice variants of At1g31090. The multiple bands could represent different isoforms with altered molecular weights.

• Proteolytic degradation: F-box proteins can be unstable. Include protease inhibitors in your extraction buffer and prepare fresh samples to minimize degradation.

• Cross-reactivity: The antibody may recognize related F-box family proteins. Perform peptide competition assays and test the antibody against recombinant proteins of related family members.

• Protein complexes: Incomplete denaturation may result in higher molecular weight complexes. Optimize SDS concentration and heating conditions.

For definitive identification of bands, consider immunoprecipitation followed by mass spectrometry analysis to confirm the identity of proteins detected by your antibody .

What strategies can help improve weak or absent At1g31090 antibody signals?

When facing weak or absent signals with an At1g31090 antibody, consider these systematic enhancement approaches:

• Sample preparation optimization:

  • Use specialized extraction buffers designed for membrane-associated proteins if At1g31090 shows membrane localization

  • Include phosphatase inhibitors if phosphorylation affects epitope recognition

  • Test different extraction methods (TCA precipitation, phenol extraction) to concentrate the protein

• Signal enhancement techniques:

  • Increase protein loading (up to 50-100 μg per lane)

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

  • Use signal amplification systems (biotin-streptavidin, tyramide signal amplification)

  • Try super-sensitive ECL substrates for Western blotting

• Epitope retrieval for immunohistochemistry:

  • Test heat-mediated (microwave, pressure cooker) or enzymatic antigen retrieval methods

  • Optimize permeabilization conditions to improve antibody accessibility

• Protein enrichment:

  • Perform subcellular fractionation to concentrate the protein in its native compartment

  • Use immunoprecipitation to concentrate the protein before Western blotting

If signals remain weak, consider whether At1g31090 is expressed at very low levels or in specific tissues/conditions only. Check transcriptomic databases to identify conditions of highest expression .

How can I minimize background when using At1g31090 antibodies in plant tissues?

High background is a common challenge when using antibodies in plant tissues due to endogenous peroxidases, phenolic compounds, and autofluorescence. For At1g31090 antibody applications, implement these strategies:

• For Western blotting:

  • Increase blocking concentration (5-10% non-fat milk or BSA)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Dilute primary antibody in blocking solution with 0.05% Tween-20

  • Include competing proteins (1-5% BSA) in antibody diluent

  • Use longer/more washes (5x 10 minutes)

• For immunohistochemistry/immunofluorescence:

  • Quench endogenous peroxidases with 0.3% H₂O₂ in methanol before blocking

  • Treat sections with 0.1M NH₄Cl to reduce autofluorescence

  • Include 0.1-0.3% Triton X-100 in blocking buffer

  • Use plant-specific blocking reagents containing non-specific plant proteins

  • Consider sodium borohydride treatment to reduce aldehyde-induced fluorescence

• General optimization:

  • Compare affinity-purified vs. whole serum antibodies

  • Test multiple secondary antibodies from different manufacturers

  • Implement more stringent washing protocols

  • Use blocking peptides to verify specificity

For Arabidopsis tissue, high levels of phenolic compounds may interfere with antibody binding. Adding polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) to extraction buffers can help sequester these compounds and reduce background .

How can I use At1g31090 antibodies to investigate protein degradation pathways?

As an F-box protein, At1g31090 likely participates in the ubiquitin-proteasome system (UPS). Antibodies against At1g31090 can be instrumental in studying these pathways:

• Characterizing SCF complex formation:

  • Perform co-immunoprecipitation with the At1g31090 antibody followed by Western blotting for known SCF components (Skp1, Cullin1, Rbx1)

  • Use proximity ligation assays (PLA) to visualize and quantify interactions in situ

  • Conduct reciprocal co-IPs with antibodies against other SCF components

• Identifying substrates:

  • Treat plants with proteasome inhibitors (MG132) to stabilize substrate proteins

  • Immunoprecipitate At1g31090 under native conditions and identify co-precipitating proteins by mass spectrometry

  • Compare ubiquitinated protein profiles between wild-type and At1g31090 knockout/knockdown plants

• Dynamics of At1g31090 expression:

  • Monitor At1g31090 protein levels across developmental stages and stress conditions

  • Investigate At1g31090 turnover using cycloheximide chase assays

  • Examine At1g31090 levels after treatment with hormones that regulate F-box protein expression

• Post-translational modifications:

  • Use phospho-specific antibodies if phosphorylation sites are known

  • Analyze At1g31090 ubiquitination status using ubiquitin antibodies after immunoprecipitation

These approaches can provide insights into how At1g31090 participates in targeted protein degradation and how this process regulates plant development or stress responses .

What are the best approaches for developing phospho-specific antibodies against At1g31090?

Developing phospho-specific antibodies against At1g31090 requires careful planning and execution:

• Identification of phosphorylation sites:

  • Analyze At1g31090 using phosphorylation prediction tools (NetPhos, PhosphoSite)

  • Perform mass spectrometry analysis of immunoprecipitated At1g31090 to identify actual phosphorylation sites

  • Check published phosphoproteomic datasets for Arabidopsis

• Peptide design considerations:

  • Design phosphopeptides containing the phosphorylated residue centrally positioned

  • Optimal length is typically 10-15 amino acids

  • Avoid hydrophobic sequences that may cause solubility issues

  • Consider adding a C-terminal cysteine for conjugation to carrier proteins

• Immunization and antibody production:

  • Immunize with the phosphopeptide conjugated to KLH or BSA

  • Use multiple rabbits to increase chances of success

  • Consider a dual immunization strategy with both phosphorylated and non-phosphorylated peptides

• Purification strategy:

  • First affinity-purify using the phosphopeptide column

  • Perform negative selection using a non-phosphopeptide column to remove antibodies recognizing the non-phosphorylated form

  • Test eluted fractions for specificity using ELISA with both phospho and non-phospho peptides

• Validation tests:

  • Western blots comparing wild-type and phosphatase-treated samples

  • Test specificity against point mutants (S/T to A) at the phosphorylation site

  • Confirm signal increase in conditions known to induce phosphorylation

The development of phospho-specific antibodies requires significant resources but can provide valuable tools for studying the regulatory mechanisms governing At1g31090 function .

How can protein microarray technology be applied for antibody screening against At1g31090?

Protein microarray technology offers powerful approaches for antibody screening and characterization:

• Antibody specificity profiling:

  • Array multiple recombinant Arabidopsis F-box proteins including At1g31090

  • Probe with the test antibody to evaluate cross-reactivity with related proteins

  • Include different domains of At1g31090 to map epitope recognition

• Antibody sensitivity assessment:

  • Array serial dilutions of recombinant At1g31090 protein (starting from ~4 fmol per spot)

  • Determine detection limits on different surfaces (FAST slides: ~2-3.6 fmol per spot; PAA slides: ~0.1-1.8 fmol per spot)

  • Compare multiple antibody lots or sources for consistency

• Epitope mapping:

  • Array overlapping peptides covering the entire At1g31090 sequence

  • Identify specific binding regions of different antibodies

  • Use competition assays with soluble peptides to confirm epitope specificity

• Cross-species reactivity testing:

  • Array homologous F-box proteins from related plant species

  • Determine antibody utility for comparative studies across species

Based on research with Arabidopsis protein chips, the detection limit for recombinant proteins can be as low as 0.1-1.8 fmol per spot on polyacrylamide slides or 2-3.6 fmol per spot on nitrocellulose-based FAST slides. For optimal results, express the full-length At1g31090 protein with an RGS-His6 tag in E. coli and purify using nickel affinity chromatography .

How should I interpret discrepancies between At1g31090 protein and mRNA expression data?

Discrepancies between protein and mRNA levels for At1g31090 are common and biologically meaningful. When faced with such differences:

• Consider post-transcriptional regulation mechanisms:

  • miRNA-mediated regulation: Check databases for predicted miRNA targeting At1g31090

  • mRNA stability differences: Measure mRNA half-life using actinomycin D treatment

  • Translation efficiency: Analyze polysome association of At1g31090 mRNA

• Examine post-translational regulation:

  • Protein stability: F-box proteins often have short half-lives; perform cycloheximide chase experiments

  • Ubiquitination: As an F-box protein, At1g31090 may undergo autoubiquitination

  • Conditional degradation: Test protein levels under various stresses or developmental stages

• Technical considerations:

  • Antibody sensitivity limits: Low-abundance proteins may be below detection threshold

  • Extraction efficiency: Membrane-associated proteins may require specialized extraction methods

  • Developmental or tissue-specific expression: Whole-tissue analysis may mask cell-specific patterns

• Experimental approach to resolve discrepancies:

  • Perform time-course experiments to capture dynamic changes

  • Use cell-type specific promoters to drive tagged versions of At1g31090

  • Implement absolute quantification methods for both mRNA (digital PCR) and protein (SRM-MS)

Remember that F-box proteins like At1g31090 often function as regulatory hubs with tightly controlled expression and may show rapid changes in response to stimuli that are not always reflected at the mRNA level .

What experimental design is optimal for studying At1g31090 protein interactions in vivo?

To comprehensively study At1g31090 protein interactions in vivo, implement a multi-layered experimental design:

• Generation of tagged At1g31090 lines:

  • Create complementation lines in at1g31090 knockout background expressing At1g31090-tag under native promoter

  • Verify functionality through phenotypic rescue

  • Consider multiple tag types: FLAG, HA, or GFP for different applications

  • Use of an inducible promoter may help overcome lethality if constitutive expression causes defects

• Verification of expression and localization:

  • Confirm expression using both tag antibodies and At1g31090-specific antibodies

  • Determine subcellular localization using confocal microscopy

  • Compare expression patterns with native At1g31090 using specific antibodies

• Interaction analysis workflow:

  • Perform standard co-IP followed by immunoblotting for suspected interaction partners

  • Conduct MS analysis of immunoprecipitates under various conditions

  • Confirm key interactions using orthogonal methods (BiFC, FRET, PLA)

  • Use crosslinking to capture transient interactions

• Temporal and spatial dynamics:

  • Study interactions across developmental stages and tissues

  • Examine changes under relevant stress conditions

  • Implement live cell imaging for real-time interaction monitoring

• Functional validation:

  • Generate mutations in interaction interfaces

  • Perform domain swapping with related F-box proteins

  • Assess phenotypic consequences of disrupted interactions

This comprehensive approach provides both identification and functional characterization of At1g31090 protein interactions, with each method compensating for limitations of others .

How can I integrate antibody-based approaches with genetic tools to study At1g31090 function?

Integrating antibody-based approaches with genetic tools creates a powerful system for functional studies:

Implementation strategies:

• Combine genetic resources with immunological tools:

  • Use T-DNA insertion lines, CRISPR/Cas9 mutants, and artificial microRNA lines targeting At1g31090

  • Verify protein expression/absence with At1g31090 antibodies

  • Create complementation lines with tagged versions for both genetic rescue and antibody detection

• Developmental and environmental studies:

  • Examine protein expression in different mutant backgrounds

  • Compare wild-type and mutant protein levels under various stresses

  • Use antibodies to track protein redistribution in response to stimuli

• Protein complex analysis:

  • Immunoprecipitate At1g31090 from various genetic backgrounds to identify context-dependent interactions

  • Compare interactome changes in related mutants

  • Analyze post-translational modifications across genetic variants

• Comparative biology:

  • Test antibody cross-reactivity with homologs in other species

  • Compare protein expression patterns across related species

  • Use antibodies to validate conserved interactions identified through comparative genomics

This integrated approach allows correlation of molecular mechanisms with phenotypic outcomes and provides a comprehensive understanding of At1g31090 function in plant biology .