At1g58090 Antibody

What is the At1g58090 gene and what is its biological significance?

The At1g58090 gene encodes a putative F-box protein that functions as part of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex in Arabidopsis thaliana. This protein plays critical roles in:

• Cellular protein ubiquitination pathways

• Regulation of protein turnover mechanisms

• Various plant development processes

As an F-box protein, At1g58090 likely contributes to phytohormone signaling and stress response pathways, though detailed functional characterization studies remain limited in the current literature. The protein has a predicted molecular weight of approximately 43 kDa, which corresponds to the band typically detected in Western blot experiments using the At1g58090 antibody.

What experimental applications is the At1g58090 antibody validated for?

The At1g58090 antibody has been validated for several experimental applications, though researchers should conduct their own validation for specific experimental conditions:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blot (WB) for identification of target antigen

What are the critical storage and handling considerations for the At1g58090 antibody?

Proper storage and handling are essential for maintaining antibody functionality:

• Upon receipt, store at -20°C or -80°C to preserve activity

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• The antibody is typically provided in liquid form with specific buffer composition:

  • Preservative: 0.03% Proclin 300

  • Constituents: 50% Glycerol, 0.01M PBS (pH 7.4)

For long-term storage planning, researchers should note the antibody is produced as "made-to-order" with a typical lead time of 14-16 weeks, requiring advance planning for experimental continuity .

How should an At1g58090 antibody be validated before use in critical experiments?

Comprehensive validation is essential before using the At1g58090 antibody in key experiments:

• Positive controls: Test against recombinant At1g58090 protein to confirm detection capability

• Negative controls: Include pre-immune serum controls to assess non-specific binding

• Knockout validation: Ideally, confirm specificity using At1g58090 knockout Arabidopsis models (though published validation using this approach is currently limited)

• Cross-reactivity assessment: Test against related F-box proteins to evaluate potential cross-reactivity

• Concentration optimization: Perform titration experiments to determine optimal antibody concentration (similar to approaches used for oligo-conjugated antibodies)

Remember that the polyclonal nature of the At1g58090 antibody preparation introduces inherent batch-to-batch variability, making validation for each new lot particularly important.

What is the optimal Western blot protocol for At1g58090 detection?

While specific protocols may require optimization for individual experimental systems, a baseline Western blot protocol for At1g58090 detection includes:

• Sample preparation:

  • Extract total protein from Arabidopsis tissue using standard extraction buffer

  • Determine protein concentration (Bradford or BCA assay)

  • Load adequate protein (typically ≥20 μg total protein) to detect the ~43 kDa target

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST (recommended blocking agent)

  • Incubate with primary At1g58090 antibody (typically 1:1000-1:2000 dilution)

  • Wash thoroughly with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection:

  • Visualize using ECL substrate and imaging system

  • Confirm band at expected molecular weight (~43 kDa)

For protein interaction studies, consider coupling with Protein A/G agarose beads for immunoprecipitation applications.

How should antibody titration be conducted to determine optimal concentrations?

Based on principles established for antibody research, a systematic titration approach is recommended:

• Initial concentration ranges:

  • Begin testing in the 0.625-2.5 μg/mL range rather than immediately using high concentrations (5-10 μg/mL)

  • This concentration range often represents the balance point between adequate signal and minimal background

• Sequential dilution series:

  • Prepare fourfold serial dilutions of the antibody

  • Test each dilution under identical experimental conditions

  • Evaluate both signal strength and signal-to-noise ratio

• Concentration-response analysis:

  • Antibodies used at concentrations ≥2.5 μg/mL often show minimal response to fourfold dilution

  • Antibodies at concentrations ≤0.62 μg/mL typically show linear response to dilution

  • The optimal concentration exists where signal reduction begins to show linear response to dilution

• Background assessment:

  • Higher concentrations often only increase background signal without improving specific detection

  • Monitor signal distribution between specific binding and background

This approach is more resource-efficient than starting with manufacturer-recommended high concentrations, which may waste reagent and create excessive background .

How can At1g58090 antibody be used in protein-protein interaction studies?

For investigating At1g58090 protein interactions with other components of the SCF complex or target substrates:

• Co-immunoprecipitation (Co-IP):

  • Immobilize At1g58090 antibody on Protein A/G agarose beads

  • Incubate with plant extract containing native protein complexes

  • Wash extensively to remove non-specific interactions

  • Elute and analyze by Western blotting for potential interaction partners

• Proximity-based labeling:

  • Consider adapting techniques similar to BioID or APEX2 proximity labeling

  • Create fusion proteins containing At1g58090 and a proximity labeling enzyme

  • Identify interacting proteins through mass spectrometry analysis

• Affinity purification with tandem mass spectrometry (AP-MS):

  • Use At1g58090 antibody for immunoprecipitation from plant extracts

  • Analyze precipitated protein complexes by LC-MS/MS

  • Identify potential binding partners and complex components

• Controls and validation:

  • Include appropriate negative controls (pre-immune serum, IgG control)

  • Confirm interactions with orthogonal methods (yeast two-hybrid, split-luciferase)

  • Validate biological relevance through functional assays

What approaches can minimize background signal when using At1g58090 antibody?

Background signal is a common challenge with antibodies. Based on principles established in antibody research, these strategies can help:

• Concentration optimization:

  • Titrate antibody to find minimum effective concentration

  • High concentrations often disproportionately increase background relative to specific signal

  • Antibodies initially used at 10 μg/mL can often be reduced substantially without compromising positive signal detection

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blockers)

  • Extend blocking time if background persists

  • Include blocking agents in antibody dilution buffer

• Stringent washing:

  • Increase number and duration of wash steps

  • Use detergent concentrations appropriate for the application

  • Consider more stringent wash buffers for high-affinity antibodies

• Pre-adsorption:

  • Pre-incubate antibody with negative control lysates to remove cross-reactive antibodies

  • Use recombinant proteins for specific pre-adsorption of cross-reactive antibodies

• Signal-to-background measurement:

  • Quantify signal distribution between specific and non-specific binding

  • For some applications, reducing antibody concentration can dramatically improve signal-to-background ratio (e.g., from 76.5% to 12.6% background)

How can At1g58090 antibody be incorporated into multimodal single-cell analysis?

Adaptation of At1g58090 antibody for cutting-edge single-cell applications requires special considerations:

What are common causes of inconsistent results when using At1g58090 antibody?

Several factors can contribute to experimental variability:

• Batch-to-batch variability:

  • Polyclonal antibody preparations inherently vary between production lots

  • Revalidate each new lot before use in critical experiments

  • Consider creating large aliquots of a single lot for longitudinal studies

• Protein extraction efficiency:

  • F-box proteins may have variable extraction efficiency based on buffer composition

  • Test multiple extraction protocols to optimize recovery

  • Ensure complete tissue disruption, particularly for plant tissues with cell walls

• Post-translational modifications:

  • F-box proteins undergo various modifications affecting antibody recognition

  • Consider phosphorylation, ubiquitination, or other modifications affecting epitope accessibility

  • Extract under conditions that preserve relevant modifications

• Protein loading consistency:

  • Ensure equal loading across samples using housekeeping protein controls

  • Quantify total protein using Bradford/BCA assays before loading

  • Consider using stain-free gel technology for loading normalization

• Detection system sensitivity:

  • F-box proteins may be low-abundance targets requiring sensitive detection

  • Match detection method sensitivity to expected protein abundance

  • Consider signal amplification for low-abundance targets

What controls are essential for interpreting At1g58090 antibody experimental data?

Comprehensive controls help ensure reliable interpretation:

Proper experimental design should include controls that allow distinguishing between true biological effects and technical artifacts.

How should researchers address signal specificity concerns with polyclonal At1g58090 antibody?

Polyclonal antibodies present specific challenges that require systematic approaches:

• Epitope mapping:

  • Identify the specific region(s) recognized by the antibody

  • Compare with homologous regions in related F-box proteins

  • Assess potential cross-reactivity through sequence alignment

• Validation in knockout/knockdown systems:

  • Generate or obtain At1g58090 knockout/knockdown Arabidopsis lines

  • Compare antibody signal between wild-type and knockout samples

  • Absence of signal in knockout samples confirms specificity

• Pre-adsorption controls:

  • Pre-incubate antibody with recombinant At1g58090 protein

  • Compare signal with and without pre-adsorption

  • Specific signal should be eliminated by pre-adsorption

• Western blot complexity analysis:

  • Evaluate number and intensity of bands detected

  • Single band at expected molecular weight suggests specificity

  • Multiple bands may indicate cross-reactivity or protein processing

• Mass spectrometry validation:

  • Immunoprecipitate with At1g58090 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm presence of target protein and assess co-precipitating proteins

How is At1g58090 antibody contributing to our understanding of plant ubiquitination pathways?

F-box proteins like At1g58090 are critical components of plant ubiquitination pathways with emerging research applications:

• Substrate identification:

  • At1g58090 antibody can help identify target proteins for ubiquitination

  • Immunoprecipitation followed by mass spectrometry enables substrate discovery

  • Understanding substrates illuminates biological function in developmental processes

• Stress response regulation:

  • F-box proteins often mediate plant responses to environmental stresses

  • At1g58090 antibody can reveal protein abundance changes during stress conditions

  • Quantitative western blotting enables temporal profiling of protein levels

• Hormone signaling integration:

  • F-box proteins frequently function in phytohormone signaling pathways

  • At1g58090 antibody can help map protein interactions with hormone signaling components

  • Co-immunoprecipitation experiments reveal regulatory networks

• Post-translational modification dynamics:

  • Antibody-based approaches can track modifications of At1g58090 itself

  • Phospho-specific antibodies could reveal regulatory modifications

  • Understanding F-box protein regulation provides systems-level insights

While At1g58090's specific biological roles remain under investigation, antibody tools enable mechanistic studies connecting this F-box protein to broader plant development pathways .

Can At1g58090 antibody be adapted for advanced microscopy applications?

Adapting At1g58090 antibody for advanced microscopy presents both challenges and opportunities:

• Immunofluorescence optimization:

  • Test fixation methods compatible with At1g58090 epitope preservation

  • Optimize permeabilization for access to subcellular compartments

  • Determine effective antibody concentration for signal-to-noise optimization

• Super-resolution microscopy adaptation:

  • Consider secondary antibody conjugates compatible with STORM/PALM

  • Evaluate direct fluorophore conjugation to minimize localization error

  • Test buffer conditions that support both antibody binding and fluorophore performance

• Multiplexed imaging approaches:

  • Combine with antibodies against known interaction partners

  • Establish spectral compatibility between fluorophores

  • Implement sequential imaging protocols for highly multiplexed detection

• Live-cell applications:

  • Explore nanobody generation against At1g58090

  • Engineer smaller antibody fragments based on heavy-chain-only antibodies

  • Test intrabody expression for live visualization of protein dynamics

Drawing from advances in nanobody technology, researchers might consider developing engineered binding proteins with enhanced membrane permeability and reduced size for improved intracellular access .

What are the challenges in applying antibody avidity engineering concepts to plant protein research?

Avidity engineering principles could enhance At1g58090 antibody performance:

• Multivalent antibody formats:

  • Creating dimeric, trimeric or higher-order antibody constructs can increase functional affinity

  • Multivalent antibodies show remarkable effectiveness in viral neutralization studies

  • Triple tandem formats have demonstrated enhanced effectiveness in other research contexts

• Implementation challenges:

  • Plant cell walls present unique barriers to antibody penetration

  • Optimization of linker length between binding domains is critical for maintaining specificity

  • Engineering must balance increased avidity against potential increases in non-specific binding

• Domain linking strategies:

  • Various formats have been used to increase valency of antibody domains:

    • Direct domain linking with optimized linker sequences

    • Fusion with dimeric Fc fragments

    • Alternative self-assembling multimerization tags

• Experimental validation:

  • BLI (Bio-Layer Interferometry) can be used to determine affinity constants

  • ELISA assays with serial dilutions help quantify avidity effects

  • Testing in plant tissues requires additional optimization for tissue penetration

Applying these concepts to plant protein research could significantly enhance detection sensitivity, particularly for low-abundance regulatory proteins like At1g58090.