At3g25460 Antibody

What is the At3g25460 protein and why is it important for Arabidopsis research?

At3g25460 (Q9LSV6) is a putative F-box protein expressed in Arabidopsis thaliana. F-box proteins are critical components of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex that mediates protein degradation through the ubiquitin-proteasome system. They play essential roles in plant development, hormone signaling, stress responses, and metabolic regulation by targeting specific proteins for degradation. The At3g25460 protein is classified as a putative F-box protein, suggesting its function in protein degradation pathways, though its precise role remains under investigation .

Understanding this protein's function through antibody-based detection methods can provide valuable insights into plant protein degradation mechanisms and their impact on various biological processes. The antibody against At3g25460 enables researchers to study protein expression patterns, localization, and interactions that would otherwise be difficult to assess through genetic approaches alone.

What are the standard applications for At3g25460 antibody in plant research?

The At3g25460 antibody has been validated for several standard applications in plant molecular biology research:

• Western Blotting (WB): The primary validated application with a recommended starting dilution of 1:1000. The antibody can detect between 0.01-1ng of its target protein in dot blot assays .

• Immunoprecipitation (IP): While specific data for At3g25460 antibody is limited, monoclonal antibodies against plant proteins can be used for immunoprecipitation studies to isolate protein complexes and study protein-protein interactions .

• ELISA: The antibody demonstrates high ELISA titer (approximately 10,000), indicating strong and specific binding to the target protein .

These applications provide researchers with tools to study protein expression levels, post-translational modifications, and protein-protein interactions involving the At3g25460 F-box protein in various plant tissues and under different experimental conditions.

How should researchers store and handle At3g25460 antibody to maintain its activity?

Proper storage and handling of the At3g25460 antibody is critical for maintaining its activity and ensuring reproducible experimental results:

• Initial handling: The antibody is supplied as a lyophilized supernatant that should be reconstituted upon delivery .

• Storage temperature: After reconstitution, store at -20°C for long-term preservation of antibody activity .

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they can lead to protein denaturation and reduced antibody activity. Aliquoting the reconstituted antibody is recommended to avoid repeated freezing and thawing .

• Working solution preparation: When preparing working dilutions, use appropriate buffer systems (typically PBS or TBS with 0.1% BSA) and prepare fresh dilutions for each experiment when possible.

• Shipping conditions: The antibody is shipped at room temperature, but should be properly reconstituted and stored immediately upon receipt .

Following these guidelines will help maintain antibody specificity and sensitivity, leading to more consistent and reliable experimental results.

What are the optimal Western blotting protocols for At3g25460 antibody?

For optimal Western blotting results with the At3g25460 antibody, researchers should consider the following methodological approach:

• Sample preparation:

  • Extract proteins from plant tissues using appropriate extraction buffers containing protease inhibitors

  • Ensure complete denaturation of proteins by heating samples in SDS loading buffer at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation of the target protein

  • Transfer proteins to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Incubate with At3g25460 antibody at a starting dilution of 1:1000 in blocking buffer overnight at 4°C

  • Wash membranes thoroughly with TBST (3-5 washes, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-mouse IgG) at 1:5000 dilution for 1 hour at room temperature

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • The antibody can detect as little as 0.01-1ng of target protein in dot blot assays, suggesting high sensitivity

Optimization may be required for specific plant tissues or experimental conditions, particularly when detecting low-abundance F-box proteins like At3g25460.

How can researchers validate the specificity of At3g25460 antibody?

Validating antibody specificity is crucial for ensuring reliable research results. For At3g25460 antibody, consider these methodological approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunogen peptide (sequences provided at shipment)

  • Run parallel Western blots with blocked and unblocked antibody

  • Disappearance of the signal in the blocked sample confirms specificity

• Genetic controls:

  • Use Arabidopsis knockout/knockdown lines for At3g25460

  • Compare antibody reactivity in wild-type vs. mutant samples

  • Absence or reduction of signal in mutant lines confirms specificity

• Recombinant protein controls:

  • Express recombinant At3g25460 protein with tags (His, GST, etc.)

  • Detect with both the At3g25460 antibody and tag-specific antibodies

  • Co-localization of signals confirms antibody specificity

• Cross-reactivity assessment:

  • Test the antibody against closely related F-box proteins

  • Minimal cross-reactivity indicates high specificity

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of At3g25460

  • Consistent results across different antibodies increase confidence in specificity

These validation methods help ensure that observed signals genuinely represent the target protein, reducing the risk of misinterpreting experimental results due to non-specific antibody binding.

What approaches can resolve detection issues with At3g25460 antibody?

When facing detection challenges with the At3g25460 antibody, researchers should consider these methodological troubleshooting approaches:

• Signal optimization strategies:

  • Adjust antibody concentration: Test a range of dilutions (1:500 to 1:5000) to find optimal signal-to-noise ratio

  • Modify incubation conditions: Try different incubation times (1 hour to overnight) and temperatures (4°C to room temperature)

  • Enhance detection sensitivity: Use signal amplification systems (biotin-streptavidin, tyramide signal amplification)

• Reducing background issues:

  • Optimize blocking conditions: Test different blocking agents (milk, BSA, commercial blockers) and concentrations

  • Increase washing stringency: Use more washing steps or higher detergent concentration

  • Add blocking agents to antibody dilution buffer: 1-5% blocking agent can reduce non-specific binding

• Protein extraction optimization:

  • Modify extraction buffers: Test different buffer compositions to improve protein solubility and stability

  • Add protease inhibitors: Prevent degradation of target proteins during sample preparation

  • Enrich for membrane proteins: If the target is membrane-associated, use specialized membrane protein extraction protocols

• Signal enhancement for low-abundance proteins:

  • Concentrate protein samples: Use immunoprecipitation to enrich the target protein before Western blotting

  • Increase sample loading: Load more total protein (50-100 μg) to detect low-abundance proteins

  • Use high-sensitivity detection reagents: Switch to femto-level ECL substrates for enhanced detection

By systematically applying these approaches, researchers can overcome common detection challenges and achieve consistent results when working with At3g25460 antibody.

How can At3g25460 antibody be used for protein-protein interaction studies?

Investigating protein-protein interactions involving the At3g25460 F-box protein is crucial for understanding its biological function. The monoclonal antibody against At3g25460 can be employed in several advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Use At3g25460 antibody to precipitate the target protein and its binding partners

  • Extract proteins under non-denaturing conditions to preserve protein-protein interactions

  • Analyze precipitated complexes by mass spectrometry to identify novel interaction partners

  • Validation can be performed by reciprocal Co-IP using antibodies against identified partners

• Proximity Ligation Assay (PLA):

  • Combine At3g25460 antibody with antibodies against suspected interaction partners

  • PLA produces fluorescent signals only when proteins are in close proximity (<40 nm)

  • This technique allows visualization of protein interactions in situ with subcellular resolution

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • While BiFC itself doesn't use antibodies, results from antibody-based interaction studies can guide BiFC experiments

  • Construct fusion proteins of At3g25460 and potential partners with split fluorescent protein fragments

  • Reconstitution of fluorescence confirms interactions identified in antibody-based studies

• Pull-down assays with SCF complex components:

  • Investigate interactions between At3g25460 and other SCF complex components (Skp1, Cullin, Rbx)

  • Use recombinant tagged components for pull-down followed by detection with At3g25460 antibody

  • This approach helps establish the role of At3g25460 in specific SCF complexes

These methodological approaches provide complementary information about protein interactions, helping to establish the biological context in which the At3g25460 F-box protein functions.

What are the considerations for immunolocalization studies using At3g25460 antibody?

Immunolocalization provides valuable information about the subcellular distribution of proteins. When using At3g25460 antibody for such studies, consider these methodological aspects:

• Tissue fixation and processing:

  • For plant tissues, use 4% paraformaldehyde fixation to preserve protein epitopes

  • Consider the need for cell wall digestion with enzymes like cellulase and pectinase

  • Optimize fixation time to balance structural preservation with antibody accessibility

• Epitope retrieval methods:

  • Heat-induced epitope retrieval may be necessary if fixation masks the epitope

  • Test different pH conditions (citrate buffer pH 6.0 vs. Tris-EDTA pH 9.0) for optimal retrieval

  • Monitor tissue integrity during retrieval to prevent sample loss

• Controls for specificity:

  • Include negative controls (primary antibody omission, non-immune IgG)

  • Use peptide competition controls with the immunogenic peptides

  • Include genetic controls (knockout/knockdown lines) when available

• Signal detection optimization:

  • Test both direct (fluorescently-labeled primary antibody) and indirect (secondary antibody) detection

  • Consider signal amplification methods for low-abundance F-box proteins

  • Use confocal microscopy for precise subcellular localization

• Co-localization studies:

  • Combine At3g25460 antibody with markers for cellular compartments (nucleus, ER, Golgi)

  • Use antibodies against other SCF complex components to verify functional localization

  • Employ quantitative co-localization analysis to assess the degree of overlap

These considerations help ensure reliable subcellular localization data, which is essential for understanding the functional context of the At3g25460 protein within plant cells.

How does titration of antibody concentration affect experimental outcomes for plant protein detection?

Antibody titration is critical for optimizing experimental conditions. Research on oligo-conjugated antibodies provides valuable insights that can be applied to plant antibodies like At3g25460:

• Balancing sensitivity and specificity:

  • Reducing antibody concentration can improve signal quality even for antibodies within their linear range

  • This approach leads to more balanced signal distribution and can reduce sequencing or detection costs

For At3g25460 antibody, these principles suggest starting at the recommended dilution (1:1000) and then conducting systematic titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background.

How does At3g25460 antibody performance compare with other plant protein antibodies?

Understanding the comparative performance of At3g25460 antibody provides valuable context for experimental design and interpretation:

• Sensitivity comparison:

  • The At3g25460 antibody can detect 0.01-1ng of its target protein in dot blot assays

  • This sensitivity is comparable to other high-quality monoclonal antibodies against plant proteins

  • For comparison, antibodies against abundant proteins like pectic homogalacturonan (LM18, LM19, LM20) typically show high sensitivity in plant tissues

• Specificity considerations:

  • As a monoclonal antibody, At3g25460 antibody offers higher specificity than polyclonal alternatives

  • The antibody is directed against specific N-terminal epitopes, potentially reducing cross-reactivity with related F-box proteins

  • Similar to other plant antibodies, specificity validation through genetic controls is highly recommended

• Application versatility:

  • Primary validation for Western blotting with potential applications in immunoprecipitation

  • Other plant antibodies, such as those targeting cell wall components, are often validated for multiple applications including immunohistochemistry

  • Expanding application range requires careful optimization and validation

• Host species considerations:

  • The At3g25460 antibody is mouse-derived (IgG) , offering compatibility with widely available secondary antibodies

  • For comparison, some plant antibodies like those against pectic homogalacturonan are rat-derived , potentially limiting secondary antibody options

This comparative analysis helps researchers contextualize the performance of At3g25460 antibody within the broader landscape of plant molecular tools, guiding expectations and experimental design.

What are the strategies for multiplexing At3g25460 antibody with other antibodies?

Multiplexing allows simultaneous detection of multiple proteins, providing valuable insights into protein relationships and cellular contexts:

• Multi-color immunofluorescence strategies:

  • Combine At3g25460 mouse monoclonal antibody with antibodies raised in different host species (rabbit, rat, goat)

  • Use species-specific secondary antibodies conjugated to different fluorophores

  • Consider spectral separation of fluorophores to minimize bleed-through during imaging

• Sequential immunoblotting approaches:

  • For Western blots, use sequential probing with antibodies of different specificities

  • Strip and reprobe membranes or use spectrally distinct detection systems

  • Consider size differences between target proteins to distinguish overlapping signals

• Addressing host species limitations:

  • When combining multiple mouse-derived antibodies, use isotype-specific secondary antibodies

  • Alternative approaches include direct labeling of primary antibodies with different fluorophores

  • Consider using fragment antibodies (Fab) to reduce cross-reactivity

• Optimization for plant tissue samples:

  • Adjust antibody concentrations individually when multiplexing to balance signal intensities

  • Control for potential cross-reactivity between antibodies and non-target plant proteins

  • Include appropriate controls to verify specificity of each antibody in the multiplex panel

• Data analysis for multiplexed experiments:

  • Use quantitative co-localization analysis for microscopy data

  • For Western blots, normalize to appropriate loading controls for each target

  • Consider computational approaches to separate overlapping signals

These methodological approaches enable researchers to obtain richer datasets by simultaneously examining At3g25460 along with other proteins of interest, providing context for its function within cellular networks.

How can researchers overcome challenges in detecting low-abundance F-box proteins like At3g25460?

F-box proteins like At3g25460 are often expressed at low levels, presenting detection challenges that require specialized approaches:

• Sample enrichment techniques:

  • Subcellular fractionation to concentrate proteins from relevant compartments

  • Immunoprecipitation or affinity purification to enrich the target protein

  • Size exclusion chromatography to separate SCF complexes containing F-box proteins

• Signal amplification methods:

  • Two-step detection systems using biotin-streptavidin for enhanced sensitivity

  • Tyramide signal amplification (TSA) for immunofluorescence applications

  • Enhanced chemiluminescence substrates with extended signal duration for Western blotting

• Protein stabilization approaches:

  • Treat samples with proteasome inhibitors (MG132) to prevent degradation of F-box proteins

  • Use phosphatase inhibitors to preserve post-translational modifications that may affect stability

  • Optimize extraction buffers to improve solubility and reduce protein aggregation

• Alternative detection technologies:

  • Mass spectrometry-based targeted proteomics for quantitative detection of specific peptides

  • Proximity ligation assay (PLA) for in situ detection with high sensitivity

  • Digital protein arrays for quantitative detection with low sample volume requirements

• Expression system considerations:

  • For biochemical studies, consider heterologous expression systems with stabilizing tags

  • Use inducible expression systems in Arabidopsis to temporarily increase protein levels

  • Co-express interaction partners that might stabilize the F-box protein

By implementing these methodological approaches, researchers can overcome the inherent challenges in studying low-abundance regulatory proteins like At3g25460, enabling more comprehensive characterization of their biological functions.

How might new antibody technologies enhance At3g25460 research?

Emerging antibody technologies offer promising avenues for advancing research on At3g25460 and other plant proteins:

• Recombinant antibody formats:

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

  • Bispecific antibodies that simultaneously target At3g25460 and potential interaction partners

  • Nanobodies derived from camelid antibodies for accessing restricted epitopes

• Site-specific conjugation techniques:

  • Controlled antibody labeling at defined positions to maintain antigen binding capacity

  • Enzyme-mediated antibody conjugation for uniform labeling with fluorophores or other detection tags

  • Click chemistry approaches for modular functionalization of antibodies

• Enhanced detection systems:

  • DNA-barcoded antibodies for highly multiplexed protein detection in single cells

  • Oligo-conjugated antibodies with optimized titration for improved signal-to-noise ratios

  • Aptamer-antibody conjugates for dual recognition of protein targets

• In vivo applications:

  • Cell-permeable antibody formats for live-cell imaging of At3g25460 dynamics

  • Optogenetic antibody systems for light-controlled manipulation of protein function

  • Antibody-based biosensors for real-time monitoring of protein activity

These technological advances could significantly enhance our ability to study the dynamics, interactions, and functions of At3g25460 in plant cellular contexts.

What considerations apply when studying At3g25460 function across different plant species?

Extending At3g25460 research beyond Arabidopsis requires careful consideration of several factors:

• Epitope conservation analysis:

  • Perform sequence alignment of At3g25460 homologs across plant species

  • Identify conserved epitopes that might be recognized by the antibody

  • Test cross-reactivity with homologs from closely related species

• Validation requirements for cross-species applications:

  • Western blot validation in each new species to confirm specificity

  • Preabsorption controls with recombinant proteins from the target species

  • Genetic controls (knockout/knockdown) when available in the target species

• F-box protein functional conservation:

  • F-box proteins often show functional diversity despite structural similarities

  • Consider potential differences in expression patterns, localization, and interaction partners

  • Correlate antibody-based findings with genetic and phenotypic data in each species

• Technical adaptations for different plant tissues:

  • Modify protein extraction protocols based on tissue composition (e.g., high secondary metabolites)

  • Adjust fixation and permeabilization for immunohistochemistry in different plant tissues

  • Optimize antibody concentration and incubation conditions for each species and tissue type

• Evolutionary context interpretation:

  • Consider evolutionary distance when interpreting cross-reactivity results

  • Use phylogenetic analysis to guide expectations for antibody performance

  • Complement antibody studies with genomic and transcriptomic comparative analyses

These considerations help researchers extend At3g25460 antibody applications beyond the model organism Arabidopsis, enabling comparative studies that provide evolutionary insights into F-box protein function.