At1g27420 Antibody

What is the At1g27420 gene and what protein does it encode?

At1g27420 is a gene locus in Arabidopsis thaliana that encodes a Galactose oxidase/kelch repeat superfamily protein. This gene is identified with the locus ID 2015979 in the Arabidopsis genome database. According to the TAIR (The Arabidopsis Information Resource) description, it belongs to the superfamily of proteins containing galactose oxidase and kelch repeat domains, suggesting potential roles in protein-protein interactions and possibly enzymatic functions related to galactose metabolism . The protein's complete functional characterization continues to be an active area of research in plant molecular biology.

What are the key specifications of commercially available At1g27420 antibodies?

Commercially available At1g27420 antibodies are typically polyclonal antibodies raised in rabbits using recombinant Arabidopsis thaliana At1g27420 protein as the immunogen. A typical example has the following specifications:

• Product Code: CSB-PA875568XA01DOA

• Host species: Rabbit

• Clonality: Polyclonal

• Reactivity: Arabidopsis thaliana

• Applications: ELISA, Western Blot (WB)

• Format: Liquid

• Storage Buffer: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS (pH 7.4)

• Purification Method: Antigen Affinity Purified

How should At1g27420 antibodies be stored and handled to maintain optimal activity?

At1g27420 antibodies require careful storage and handling to maintain their specificity and activity. The recommended storage conditions are:

• Store at -20°C or -80°C upon receipt

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

• For lyophilized formats, reconstitute by adding the recommended volume of sterile water (typically 50 μl)

• After reconstitution, make aliquots to minimize freeze-thaw cycles

• Briefly centrifuge tubes before opening to avoid loss of material adhering to the cap or sides

• For shipping purposes, the product is typically transported at 4°C but should be stored immediately at the recommended temperature upon receipt

What are the validated applications for At1g27420 antibodies in plant research?

At1g27420 antibodies have been validated for several experimental applications in plant molecular biology research:

• Western Blotting (WB): The primary application, typically using dilutions of 1:2500 to 1:5000. This technique allows researchers to detect the presence and relative abundance of the target protein in plant tissue extracts.

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative determination of protein levels in solution.

• Immunohistochemistry: Although less commonly reported, some antibodies may be suitable for localization studies in fixed tissue sections.

For Western blot applications, researchers should denature protein samples (typically 10 μg of total protein) using appropriate sample buffers at 70°C for 5 minutes before separation on SDS-PAGE gels. After transfer to PVDF membranes, blocking with 5% milk in TBS-T for 1 hour at room temperature is recommended before incubation with the primary antibody .

What is the recommended protocol for Western blotting using At1g27420 antibodies?

For optimal Western blot results with At1g27420 antibodies, the following protocol is recommended:

• Sample preparation:

  • Extract total protein from Arabidopsis thaliana tissues

  • Store extracted proteins at -80°C until use

  • Denature 10 μg of total protein with LDS sample buffer (4X) at 70°C for 5 minutes

• Electrophoresis and transfer:

  • Separate proteins on a Bis-Tris 4-12% SDS-PAGE gel

  • Transfer to PVDF membrane (0.45 μm pore size) using wet transfer for 1 hour

• Antibody incubation:

  • Block membrane with 5% milk in TBS-T for 1 hour at room temperature with agitation

  • Incubate with primary antibody at 1:2000 dilution for 1 hour at room temperature

  • Wash membrane thoroughly with TBS-T

  • Incubate with appropriate secondary antibody (anti-rabbit IgG) conjugated to a detection system

• Detection:

  • Develop using chemiluminescence or other appropriate detection methods

  • The expected molecular weight for the At1g27420 protein is approximately 28 kDa, though the apparent molecular weight may vary slightly based on post-translational modifications

How can researchers determine the optimal antibody dilution for their specific experimental conditions?

Determining the optimal antibody dilution requires systematic titration experiments:

• Prepare a dilution series of the primary antibody (e.g., 1:1000, 1:2000, 1:5000, 1:10000)

• Run identical Western blots with these different dilutions, keeping all other variables constant

• Evaluate results based on:

  • Signal-to-noise ratio (higher is better)

  • Background levels (lower is better)

  • Specificity (presence of single band at expected molecular weight)

  • Signal intensity (sufficient for detection but not oversaturated)

For At1g27420 antibodies, starting with the manufacturer's recommended dilution range (1:2500-1:5000 for Western blot) is advisable, then adjusting based on your specific tissue type, protein abundance, and detection system sensitivity. Some researchers report successful results with dilutions as low as 1:2000 for plant tissue extracts when using enhanced chemiluminescence detection systems .

How do At1g27420 antibodies perform across different plant species and what cross-reactivity should researchers be aware of?

Cross-reactivity is an important consideration when working with plant antibodies. While At1g27420 antibodies are specifically designed for Arabidopsis thaliana, they may show reactivity with homologous proteins in closely related species. Based on sequence homology and experimental validation:

• Confirmed reactivity: Primarily with Arabidopsis thaliana

• Predicted reactivity based on sequence homology:

  • Brassica species (e.g., Brassica napus)

  • Other members of the Brassicaceae family

• No reactivity expected in:

  • Monocot species like Oryza sativa (rice)

  • Non-plant organisms

When working with species other than Arabidopsis, researchers should perform preliminary validation experiments to confirm antibody specificity and adjust protocols accordingly. Western blotting with positive controls (Arabidopsis extracts) alongside your species of interest can help establish cross-reactivity .

What are the challenges in interpreting At1g27420 antibody results when studying protein-protein interactions?

Interpreting antibody results in protein-protein interaction studies presents several challenges:

• Epitope masking: Protein interactions may obscure antibody binding sites, leading to false negatives. This is particularly relevant for kelch repeat proteins like At1g27420, which function through protein-protein interactions.

• Co-immunoprecipitation considerations:

  • Use gentler lysis conditions to preserve protein complexes

  • Consider crosslinking approaches to stabilize transient interactions

  • Validate results with reciprocal co-IP experiments using antibodies against predicted interaction partners

• Competition with endogenous proteins: When performing pull-down assays, be aware that endogenous proteins may compete with tagged recombinant proteins.

• Specificity validation: Always include appropriate negative controls and validate interactions using orthogonal methods such as yeast two-hybrid or bimolecular fluorescence complementation.

Given that At1g27420 encodes a Galactose oxidase/kelch repeat superfamily protein, it likely participates in protein complexes that could affect antibody accessibility to the target epitope .

How can researchers address non-specific binding issues with At1g27420 antibodies?

Non-specific binding is a common challenge with polyclonal antibodies. To address this issue:

• Optimization strategies:

  • Increase blocking agent concentration (e.g., BSA or milk from 3% to 5%)

  • Extend blocking time (from 1 hour to overnight at 4°C)

  • Implement more stringent washing (increase number of washes and duration)

  • Add low concentrations of detergent (0.1-0.3% Tween-20) to washing buffer

  • Pre-adsorb antibody with plant extract from knockout mutants

• Validation approaches:

  • Include negative controls such as extracts from at1g27420 knockout/knockdown lines

  • Compare with secondary antibody-only controls to identify non-specific bands

  • Test specificity by peptide competition assays

• Alternative detection methods:

  • Consider using fluorescent secondary antibodies which often provide better signal-to-noise ratios than enzyme-based detection systems

  • Use two-color detection systems to better distinguish non-specific binding

If persistent non-specific binding occurs, immunoprecipitation followed by mass spectrometry can help identify cross-reacting proteins .

What are the critical factors in designing experiments to study At1g27420 gene function using antibodies?

When designing experiments to study At1g27420 function using antibodies, researchers should consider:

• Experimental controls:

  • Positive control: Arabidopsis wild-type samples with known expression of At1g27420

  • Negative control: at1g27420 knockout/knockdown mutants

  • Loading control: Antibodies against constitutively expressed proteins (e.g., actin)

• Developmental considerations:

  • At1g27420 may show tissue-specific or developmental stage-specific expression

  • Include a developmental series to capture potential temporal regulation

  • Compare different tissues to identify spatial expression patterns

• Environmental factors:

  • Consider testing plants grown under different conditions (light, temperature, stress)

  • Include appropriate time-course experiments for environmentally responsive expression

• Genetic background:

  • Use consistent ecotypes to avoid natural variation confounding results

  • Consider complementation studies with tagged versions to confirm antibody specificity

• Technical considerations:

  • Optimize protein extraction methods for your specific tissue type

  • Consider native vs. denaturing conditions depending on your research question

  • Include appropriate sample replication for statistical validity

What troubleshooting approaches are effective when At1g27420 antibody detection yields weak or no signal?

When facing weak or absent signals with At1g27420 antibodies, systematic troubleshooting is essential:

• Sample preparation issues:

  • Verify protein extraction efficiency using total protein stains

  • Test alternative extraction buffers with different detergents

  • Ensure protease inhibitors are fresh and appropriate for plant tissues

  • Check protein concentration and loading consistency

• Technical adjustments:

  • Decrease antibody dilution (e.g., from 1:5000 to 1:2000)

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

  • Use enhanced sensitivity detection systems (enhanced chemiluminescence)

  • Optimize transfer conditions (longer transfer time for larger proteins)

  • Switch membrane type (PVDF vs. nitrocellulose)

• Biological considerations:

  • Verify expression levels of At1g27420

  • Consider protein degradation or post-translational modifications

  • Test different tissues or developmental stages where expression may be higher

  • Examine expression under conditions that may induce the gene/protein

• Antibody quality assessment:

  • Check antibody age and storage conditions

  • Verify with a small-scale dot blot test before full experiment

  • Consider using a new antibody batch if available

How should researchers interpret contradictory results between antibody-based detection methods and transcriptomic data for At1g27420?

Discrepancies between protein detection and transcriptomic data are common in biological research and require careful interpretation:

• Potential biological explanations:

  • Post-transcriptional regulation (miRNA targeting, RNA stability)

  • Translational control (ribosome occupancy, translation efficiency)

  • Post-translational regulation (protein stability, degradation pathways)

  • Protein localization or compartmentalization affecting extraction efficiency

• Technical considerations:

  • Sensitivity differences between techniques (RNA-seq vs. Western blot)

  • Antibody specificity issues (cross-reactivity with related proteins)

  • Limitations in protein extraction efficiency from certain tissues

  • Temporal differences (transcript levels change more rapidly than protein levels)

• Validation approaches:

  • Perform qRT-PCR to validate transcriptomic data

  • Use tagged versions of At1g27420 for independent detection

  • Employ multiple antibodies targeting different epitopes

  • Consider proteomics approaches for independent protein quantification

• Experimental design adjustments:

  • Include time-course experiments to capture delayed protein expression

  • Compare different extraction methods to ensure complete protein recovery

  • Use fractionation approaches to examine protein localization effects

These discrepancies often reveal important regulatory mechanisms and should be viewed as research opportunities rather than technical failures .

How can researchers integrate antibody-based techniques with other methods to comprehensively study At1g27420 function?

A multi-method approach provides the most comprehensive understanding of At1g27420 function:

• Integrating transcriptomics and proteomics:

  • Combine RNA-seq data with antibody-based protein quantification

  • Correlate transcript and protein levels across developmental stages

  • Identify conditions where post-transcriptional regulation may occur

• Functional genomics integration:

  • Use antibodies to validate knockout/knockdown efficiency at protein level

  • Employ ChIP-seq with transcription factor antibodies to identify regulators of At1g27420

  • Combine with metabolomics to link protein function to metabolic pathways

• Protein interaction studies:

  • Use antibodies for co-immunoprecipitation followed by mass spectrometry

  • Validate interactions with techniques like bimolecular fluorescence complementation

  • Employ proximity labeling techniques (BioID, APEX) with antibody validation

• Subcellular localization:

  • Combine immunofluorescence with cell fractionation and Western blotting

  • Use super-resolution microscopy with antibody labeling

  • Correlate with fluorescent protein fusion localization patterns

This integrated approach leverages the strengths of antibody-based detection while compensating for potential limitations through independent validation methods .

What are the advanced immunoprecipitation protocols optimized for plant kelch repeat proteins like At1g27420?

Advanced immunoprecipitation protocols for kelch repeat proteins require special considerations:

• Optimized extraction buffer components:

  • Non-ionic detergents (0.5-1% NP-40 or Triton X-100)

  • Moderate salt concentration (150-300 mM NaCl)

  • Glycerol (10%) to stabilize protein-protein interactions

  • Comprehensive protease inhibitor cocktail

  • Phosphatase inhibitors if studying phosphorylation events

• Specialized IP techniques:

  • Tandem affinity purification for improved purity

  • Formaldehyde crosslinking to capture transient interactions

  • Sequential immunoprecipitation to identify specific complexes

  • On-bead digestion for direct mass spectrometry analysis

• Protocol modifications for kelch repeat proteins:

  • Gentler lysis conditions to preserve native protein complexes

  • Shorter incubation times to minimize dissociation of complexes

  • Addition of specific cofactors that may stabilize interactions

  • Pre-clearing lysates with protein A/G beads to reduce background

• Controls and validation:

  • IgG control immunoprecipitations

  • Use of knockout/knockdown lines as negative controls

  • Reciprocal co-IPs to confirm interactions

  • Competition with excess antigen to demonstrate specificity

The kelch repeat domains in the At1g27420 protein likely mediate protein-protein interactions, making these optimizations particularly important for functional studies .

How can researchers quantitatively analyze At1g27420 protein expression across different experimental conditions?

Quantitative analysis of At1g27420 protein requires rigorous methodological approaches:

• Western blot quantification methods:

  • Densitometry analysis with appropriate normalization to loading controls

  • Use of standard curves with recombinant protein for absolute quantification

  • Implementation of digital image acquisition systems with linear detection ranges

  • Statistical analysis across multiple biological replicates (n≥3)

• ELISA-based quantification:

  • Development of sandwich ELISA using At1g27420 antibodies

  • Creation of standard curves using purified recombinant protein

  • Analysis of multiple dilutions to ensure measurements in linear range

  • Inclusion of spike-in controls to assess matrix effects

• Quantitative mass spectrometry approaches:

  • Label-free quantification comparing peptide intensities

  • Stable isotope labeling (SILAC, TMT, iTRAQ) for multiplexed comparison

  • Selected reaction monitoring (SRM) for targeted quantification

  • Standard addition methods for absolute quantification

• Data analysis and presentation:

  • Appropriate statistical tests (e.g., ANOVA with post-hoc tests)

  • Clear representation of biological and technical variability

  • Normalization to account for extraction efficiency differences

  • Reporting of both absolute and relative quantification where possible

The combination of these approaches provides robust quantitative assessment of At1g27420 protein levels across experimental conditions, tissues, or genotypes .