At4g26350 Antibody

What is At4g26350 and why is it significant in plant research?

At4g26350 refers to a specific gene locus in Arabidopsis thaliana that encodes an F-box/RNI-like/FBD-like domains-containing protein . This protein belongs to the diverse F-box protein family, which plays crucial roles in ubiquitin-mediated protein degradation pathways in plants. F-box proteins are components of SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase complexes that regulate numerous cellular processes including hormonal responses, developmental pathways, and stress responses. The study of At4g26350 contributes to our understanding of plant protein regulation and signal transduction mechanisms. Antibodies against this protein enable researchers to investigate its expression patterns, subcellular localization, and potential protein-protein interactions.

What techniques can I use to validate an At4g26350 antibody before experimental application?

Antibody validation is crucial for ensuring experimental integrity. For At4g26350 antibody validation, implement a multi-step approach:

• Genetic controls: Test antibody in wild-type versus At4g26350 knockout/knockdown lines to confirm specificity

• Western blot analysis: Verify single band of expected molecular weight (~predicted size may vary from calculated MW)

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm epitope specificity

• Cross-reactivity testing: Evaluate against closely related F-box proteins

• Immunoprecipitation followed by mass spectrometry: Confirm target identity

Validation remains an underdeveloped practice despite its importance, with researchers citing time constraints and cost as significant barriers . Document all validation steps thoroughly in your methods section to promote research reproducibility.

How does At4g26350 antibody specificity affect experimental design?

The specificity of an At4g26350 antibody directly influences experimental design decisions. Most commercial At4g26350 antibodies are polyclonal, raised in rabbits against recombinant Arabidopsis thaliana At4g26350 protein . This has several implications:

• Epitope consideration: Polyclonal antibodies recognize multiple epitopes, which can increase sensitivity but may also increase cross-reactivity

• Application suitability: While labeled for ELISA and WB applications , each detection method requires separate validation

• Sample preparation protocol: Different extraction buffers may expose different epitopes

• Batch variation management: Include consistent positive controls across experiments due to potential lot-to-lot variability in polyclonal preparations

Researchers must design experiments acknowledging these characteristics, particularly when quantitative comparisons across different studies are needed.

What are the optimal protocols for using At4g26350 antibody in Western blotting?

For optimal Western blot results with At4g26350 antibody, follow this detailed protocol:

• Sample preparation:

  • Extract total protein from Arabidopsis tissue in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Centrifuge at 14,000×g for 15 minutes at 4°C and collect supernatant

  • Quantify protein concentration using Bradford assay

• Gel electrophoresis and transfer:

  • Load 20-30μg protein per lane on 10-12% SDS-PAGE gel

  • Separate proteins at 120V for 90 minutes

  • Transfer to PVDF membrane (0.45μm) at 100V for 60 minutes in cold transfer buffer

• Immunoblotting:

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

  • Incubate with At4g26350 antibody at 1:500-1:2,000 dilution in blocking buffer overnight at 4°C

  • Wash 3×10 minutes with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5,000) for 1 hour

  • Wash 3×10 minutes with TBST

  • Develop using ECL substrate and image

• Controls:

  • Include positive control (wild-type Arabidopsis extract)

  • Include negative control (At4g26350 knockout line extract if available)

  • Molecular weight marker to confirm target size

The apparent protein size on Western blot may differ from calculated molecular weight due to post-translational modifications or protein structure .

How can I optimize immunolocalization experiments using At4g26350 antibody?

For successful immunolocalization of At4g26350 in plant tissues:

• Tissue fixation and embedding:

  • Fix freshly harvested tissue in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at 4°C

  • Dehydrate through ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Embed in paraffin or optimal cutting temperature (OCT) compound for cryosectioning

• Sectioning and antigen retrieval:

  • Cut 8-10μm sections

  • For paraffin sections: deparaffinize with xylene and rehydrate

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

• Immunostaining:

  • Block with 2% BSA, 5% normal goat serum in PBS for 1 hour

  • Incubate with At4g26350 antibody (1:100-1:500) overnight at 4°C

  • Wash 3×10 minutes with PBS

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour

  • Counterstain nuclei with DAPI

  • Mount and image using confocal microscopy

• Critical controls:

  • Primary antibody omission

  • Pre-immune serum control

  • Peptide competition control

  • Genetic controls (comparing wild-type to knockout lines)

When interpreting results, compare subcellular localization patterns with other characterized F-box proteins, using approaches similar to those employed for studying nuclear localization of other plant proteins .

What considerations are important when using At4g26350 antibody in co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments with At4g26350 antibody:

• Buffer optimization:

  • Use gentle lysis buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40, 10% glycerol) with freshly added protease inhibitors

  • Avoid harsh detergents that may disrupt protein-protein interactions

• Antibody conjugation:

  • Consider direct conjugation to magnetic beads to minimize background

  • Alternatively, use Protein A/G beads for rabbit polyclonal antibodies

  • Determine optimal antibody:bead ratio (typically 2-5μg antibody per 50μl bead slurry)

• Experimental workflow:

  • Pre-clear lysate with naked beads to reduce non-specific binding

  • Incubate cleared lysate with antibody-conjugated beads (4°C, 3-4 hours)

  • Wash extensively (at least 5 times) with reducing detergent concentration

  • Elute bound proteins with SDS sample buffer or gentle elution buffer

• Validation approaches:

  • Perform reverse co-IP with antibodies against suspected interaction partners

  • Include IgG control

  • Confirm specificity through mass spectrometry analysis

  • Validate interactions with orthogonal methods (Y2H, BiFC)

When studying potential interactions of At4g26350 with other proteins, consider that F-box proteins typically interact with SKP1 and other SCF complex components. Look for interactions similar to those observed between other plant proteins like ATG6 and NPR1, where direct protein-protein interactions have been successfully demonstrated .

What are common issues when using At4g26350 antibody and how can they be resolved?

Researchers frequently encounter issues with antibody specificity, with many commercial antibodies recognizing additional unintended molecules, compromising research integrity . When troubleshooting, always revisit validation data and consider re-validation if results are inconsistent.

How can I address batch-to-batch variability in At4g26350 antibody performance?

Batch-to-batch variability is a significant concern with polyclonal antibodies like those raised against At4g26350. To manage this variability:

• Validation strategy:

  • Re-validate each new antibody lot before use in critical experiments

  • Perform side-by-side comparison with previous lot using identical samples

  • Document lot-specific optimal working dilutions

• Reference standards:

  • Maintain a reference sample set (positive control) stored in single-use aliquots at -80°C

  • Use these standards to calibrate new antibody batches

  • Consider creating a standard curve for quantitative applications

• Experimental design adaptations:

  • Complete experimental series with a single antibody lot when possible

  • If lot changes are unavoidable mid-experiment, include overlapping samples

  • Document lot numbers in methods sections of publications

• Documentation practices:

  • Maintain detailed records of antibody performance by lot

  • Record optimization parameters for each application

  • Share this information with collaborators

Recognizing that batch variability is an inherent property of biological reagents like antibodies , implement these practices to reduce its impact on research reproducibility.

What strategies can improve detection sensitivity when working with low-abundance At4g26350 protein?

When studying low-abundance proteins like At4g26350:

• Sample preparation optimization:

  • Enrich for nuclear fraction (where many F-box proteins function)

  • Use phosphatase inhibitors to preserve modified forms

  • Consider subcellular fractionation to concentrate target protein

• Signal amplification methods:

  • Employ TSA (Tyramide Signal Amplification) for immunohistochemistry

  • Use high-sensitivity ECL substrate for Western blots

  • Consider biotin-streptavidin amplification systems

• Protein concentration techniques:

  • Immunoprecipitate target protein before detection

  • Use TCA precipitation to concentrate proteins from dilute samples

  • Consider using plant tissues/conditions with higher expression

• Detection system selection:

  • Choose fluorescent secondary antibodies with appropriate spectral properties

  • Use cooled CCD camera systems for chemiluminescence detection

  • Consider LI-COR infrared detection systems for quantitative applications

When working with low-abundance proteins, validation becomes even more critical, as the risk of detecting non-specific signals increases . Always confirm results with complementary approaches such as transcript analysis or tagged protein expression.

How can I use At4g26350 antibody to study protein degradation dynamics?

F-box proteins like At4g26350 are often involved in protein degradation pathways. To study these dynamics:

• Protein stability assays:

  • Perform cycloheximide chase experiments to track protein degradation rates

  • Compare substrate protein levels in wild-type vs. At4g26350 mutant backgrounds

  • Use proteasome inhibitors (MG132) to confirm ubiquitin-proteasome involvement

• Ubiquitination detection:

  • Immunoprecipitate potential substrate proteins

  • Probe with anti-ubiquitin antibodies

  • Compare ubiquitination patterns in wild-type vs. At4g26350 mutant plants

• Interaction kinetics analysis:

  • Use biolayer interferometry to measure binding kinetics between At4g26350 and potential substrates

  • Determine association/dissociation rates (kon/koff)

  • Compare binding affinity (KD) under different conditions

• In vivo degradation visualization:

  • Generate fluorescent protein fusions with potential substrates

  • Monitor fluorescence intensity changes over time

  • Compare degradation kinetics in wild-type vs. At4g26350 mutant backgrounds

This approach parallels methods used to study other protein degradation systems in plants, such as the proteasome-dependent proteolysis observed with MYC2 .

What approaches can integrate At4g26350 antibody with systems biology studies?

To incorporate At4g26350 research into broader systems biology frameworks:

• Protein interaction network mapping:

  • Use At4g26350 antibody for immunoprecipitation coupled with mass spectrometry

  • Identify interaction partners under different conditions

  • Validate key interactions with techniques like BiFC or FRET

  • Construct interaction networks using computational tools

• Multi-omics integration:

  • Correlate At4g26350 protein levels with transcriptome data

  • Compare proteome changes in wild-type vs. At4g26350 mutants

  • Integrate with metabolomics to identify pathway impacts

  • Use network analysis to identify regulatory modules

• Temporal and spatial profiling:

  • Apply At4g26350 antibody in tissue-specific Western blots

  • Perform immunohistochemistry across developmental stages

  • Create protein expression maps in response to stimuli

  • Correlate with tissue-specific transcriptome data

• Computational modeling:

  • Use quantitative At4g26350 protein data to parameterize models

  • Simulate F-box protein network dynamics

  • Predict system responses to perturbations

  • Validate model predictions experimentally

This integrated approach follows principles similar to those used in studying complexes like ATG6-NPR1, where protein interactions lead to functional synergy in plant immunity .

How can I leverage At4g26350 antibody in gene editing validation studies?

For CRISPR/Cas9 or other gene editing approaches targeting At4g26350:

• Protein-level validation strategy:

  • Use Western blot with At4g26350 antibody to confirm knockout

  • Compare protein levels in wild-type, heterozygous, and homozygous edited lines

  • Detect truncated proteins resulting from frameshift mutations

  • Validate multiple independent edited lines

• Epitope consideration in editing design:

  • Map antibody epitope region on At4g26350 protein

  • Consider designing edits that eliminate epitope recognition

  • Alternatively, preserve epitope for validation purposes

  • Use epitope information to predict if truncated proteins will be detectable

• Quantitative assessment workflow:

  • Perform quantitative Western blot using standard curves

  • Compare At4g26350 protein levels across edited lines

  • Correlate protein reduction with phenotypic changes

  • Use immunohistochemistry to verify tissue-specific editing efficiency

• Functional validation approach:

  • Combine protein detection with interaction studies

  • Assess if edited protein maintains binding to known partners

  • Evaluate subcellular localization changes

  • Test pathway functionality through substrate degradation assays

When evaluating edited lines, consider that antibody-based validation provides complementary information to genomic sequencing, offering direct evidence of protein-level changes resulting from gene editing.

How might new antibody technologies enhance At4g26350 research?

Emerging antibody technologies offer new possibilities for At4g26350 research:

• Nanobody development:

  • Single-domain antibodies with smaller size (~15 kDa)

  • Enhanced tissue penetration for in vivo imaging

  • Potential for intrabody applications

  • Greater stability under varying conditions

• Proximity labeling applications:

  • At4g26350 antibody conjugated to enzymes like APEX2 or BioID

  • Enables spatial proteomics to identify proximal proteins

  • Maps microenvironments where At4g26350 functions

  • Identifies transient interactions difficult to capture by co-IP

• Super-resolution microscopy compatibility:

  • Site-specific labeling with small fluorophores

  • Reduced linkage error for precise localization

  • Compatible with techniques like STORM or PALM

  • Enables visualization of protein nanoclusters

• Recombinant antibody fragments:

  • Defined specificity with reduced batch variation

  • Engineered affinity for specific applications

  • Potential for multiplexed detection strategies

  • Humanized versions for in vivo applications

These technological advances parallel the development of engineered antibodies in other research areas, where techniques like yeast surface display have been used to optimize binding properties .

What interdisciplinary approaches could benefit from At4g26350 antibody use?

At4g26350 antibody applications extend beyond traditional plant molecular biology:

• Synthetic biology integration:

  • Engineer synthetic ubiquitin ligase systems based on At4g26350

  • Create tunable protein degradation switches

  • Design orthogonal signaling pathways

  • Antibody used to validate synthetic circuit function

• Agricultural biotechnology applications:

  • Study At4g26350 role in stress responses and growth regulation

  • Develop crops with modified F-box protein networks

  • Antibody used to monitor protein expression in transgenic lines

  • Compare protein conservation across crop species

• Evolutionary biology perspectives:

  • Compare At4g26350 expression patterns across plant species

  • Study functional conservation of F-box proteins

  • Evaluate epitope conservation in antibody cross-reactivity tests

  • Reconstruct evolutionary history of F-box protein functions

• Computational biology integration:

  • Use antibody-derived protein quantification for model parameterization

  • Predict protein interaction networks

  • Simulate cell signaling dynamics

  • Validate computational predictions with antibody-based assays

These approaches draw inspiration from interdisciplinary studies like those examining plant immunity mechanisms, where protein interactions contribute to network-level understanding of biological processes .

How can I contribute to improving antibody validation standards for plant research?

Researchers can advance antibody validation standards by:

• Implementation of validation frameworks:

  • Adopt multi-pillar validation approaches

  • Document all validation experiments thoroughly

  • Share validation data through repositories

  • Include detailed validation methods in publications

• Community resource development:

  • Contribute to plant antibody databases

  • Share protocols and optimization parameters

  • Participate in multi-laboratory validation studies

  • Report issues with commercial antibodies to vendors

• Education and training initiatives:

  • Train junior researchers in validation best practices

  • Develop standardized validation protocols for plant antibodies

  • Create educational resources on antibody validation

  • Address common misconceptions about antibody specificity

• Publication practices improvement:

  • Include comprehensive antibody information (catalog numbers, lots, dilutions)

  • Publish validation data as supplementary material

  • Cite relevant validation studies

  • Be transparent about limitations and failures

These efforts align with broader initiatives to improve research reproducibility, addressing key behavioral drivers of antibody validation problems, including time constraints, cost concerns, and lack of standardized approaches .