AGL14 Antibody

What is AGL14 Antibody and what organism does it target?

AGL14 Antibody is a polyclonal antibody raised against the AGAMOUS-LIKE 14 (AGL14) protein, a MADS-box transcription factor from Arabidopsis thaliana (Mouse-ear cress). The antibody specifically recognizes the recombinant Arabidopsis thaliana AGL14 protein and is produced in rabbits through immunization with purified recombinant protein . This antibody is designed for detecting endogenous AGL14 protein in plant tissue samples and is primarily reactive with Arabidopsis thaliana samples. The fundamental role of this antibody is to enable researchers to study AGL14 protein expression, localization, and function in plant developmental biology.

What are the validated applications for AGL14 Antibody?

AGL14 Antibody has been validated for several research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative measurement of AGL14 protein levels

• Western Blotting (WB) - For detection of AGL14 protein in complex mixtures and assessment of protein expression levels

These applications have been validated to ensure specific identification of the antigen . While these represent the confirmed applications, researchers should conduct preliminary validation tests when adapting the antibody for other immunological techniques such as immunohistochemistry or immunoprecipitation.

What are the optimal storage conditions for maintaining AGL14 Antibody activity?

To maintain optimal activity of AGL14 Antibody, the following storage conditions are recommended:

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

• Avoid repeated freeze-thaw cycles that can lead to protein denaturation and loss of activity

• The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4

For long-term storage, -80°C is preferable, while -20°C is suitable for antibodies that will be used within several months. When working with the antibody, aliquoting into smaller volumes before freezing is recommended to avoid multiple freeze-thaw cycles.

What optimization strategies should be employed when using AGL14 Antibody for Western blotting?

When optimizing Western blotting protocols with AGL14 Antibody, consider the following methodological approaches:

Sample Preparation:

• Use fresh plant tissue samples whenever possible

• Include protease inhibitors during extraction to prevent protein degradation

• Optimize protein extraction buffers for nuclear proteins (as AGL14 is a transcription factor)

Blotting Parameters:

• Test dilution ranges between 1:500 to 1:2000 for primary antibody

• Optimize blocking conditions (5% non-fat milk or BSA)

• Consider longer incubation times (overnight at 4°C) for improved sensitivity

Detection System:

• For low abundance proteins, chemiluminescent detection systems offer higher sensitivity

• Consider signal enhancement systems when working with tissues where AGL14 expression is limited

Controls:

• Include positive controls (Arabidopsis thaliana wild-type tissue)

• Use negative controls (AGL14 knockout lines if available)

• Include loading controls (anti-actin or anti-histone antibodies)

As this antibody is affinity-purified , it typically provides good specificity, but background optimization may still be necessary depending on your specific tissue type and extraction method.

How can researchers design experiments to validate antibody specificity for AGL14 protein?

Validating antibody specificity is crucial for generating reliable data. For AGL14 Antibody, consider these validation approaches:

Genetic Validation:

• Compare antibody detection in wild-type plants versus AGL14 knockout/knockdown lines

• Use overexpression lines to confirm increased signal intensity

Molecular Validation:

• Perform peptide competition assays by pre-incubating the antibody with excess recombinant AGL14 protein

• Conduct immunoprecipitation followed by mass spectrometry to confirm the identity of pulled-down proteins

Functional Validation:

• Correlate protein detection with known expression patterns of AGL14 mRNA

• Compare results with alternative antibodies against the same target (if available)

Technical Replication:

• Test specificity across multiple technical platforms (ELISA, WB, etc.)

• Validate across different tissue types and developmental stages

Document all validation steps thoroughly in your methodology to strengthen the reliability of your research findings.

What approaches should be considered when adapting AGL14 Antibody for chromatin immunoprecipitation (ChIP) experiments?

While AGL14 Antibody has not been explicitly validated for ChIP applications , researchers interested in using it for chromatin studies should consider:

Protocol Adaptation:

• Optimize crosslinking conditions (1-2% formaldehyde for 10-15 minutes)

• Test sonication parameters to achieve DNA fragments of 200-500 bp

• Use higher antibody concentrations (2-5 µg per reaction) for initial optimization

Controls for ChIP Validation:

• Include input chromatin control

• Use IgG from rabbit serum as a negative control

• Target known AGL14 binding sites from literature for positive control regions

Antibody-Specific Considerations:

• Pre-clear chromatin with protein A/G beads to reduce background

• Extend incubation time (overnight at 4°C with rotation)

• Consider dual crosslinking (formaldehyde plus protein-specific crosslinkers)

Validation Approach:

• Start with qPCR of predicted binding regions

• Confirm enrichment at known MADS-box binding motifs (CArG boxes)

• Proceed to ChIP-seq only after successful validation by qPCR

As a polyclonal antibody, batch-to-batch variation might affect ChIP efficiency, so preliminary testing with each new lot is recommended.

How should researchers analyze contradictory results between AGL14 protein detection and gene expression data?

When faced with discrepancies between protein levels detected with AGL14 Antibody and corresponding mRNA expression data, consider the following analytical framework:

Possible Explanations for Contradictions:

Resolution Approaches:

• Perform time-course experiments to capture temporal dynamics

• Use cell-type specific markers alongside AGL14 detection

• Employ protein degradation inhibitors to assess protein turnover rates

• Confirm results with alternative detection methods (e.g., mass spectrometry)

• Analyze regulation by miRNAs or other post-transcriptional regulators

When reporting such contradictions, present both datasets transparently and discuss possible biological mechanisms rather than dismissing either result as experimental error.

What considerations should guide quantitative analysis of AGL14 protein expression across different developmental stages?

Quantitative analysis of AGL14 protein expression requires careful experimental design and data normalization:

Experimental Design Considerations:

• Sample developmental stages systematically (establish clear morphological markers)

• Include biological replicates (minimum n=3) for each developmental stage

• Process all samples simultaneously to minimize technical variation

Normalization Strategies:

• Use constitutively expressed proteins (actin, tubulin) as loading controls

• Consider developmental stage-specific reference proteins when appropriate

• Employ total protein normalization methods (Stain-Free technology, Ponceau S)

Quantification Methods:

• Use densitometry software with background subtraction

• Establish linear range of detection for antibody

• Report relative expression ratios rather than absolute values

• Include statistical analysis (ANOVA with post-hoc tests) when comparing stages

Data Presentation:

• Present normalized data with error bars

• Include representative Western blot images

• Document image acquisition parameters and software used for quantification

This methodical approach helps ensure reproducibility and reliable interpretation of developmental expression patterns.

How can researchers address weak or absent signal when using AGL14 Antibody?

When encountering weak or absent signals with AGL14 Antibody, employ this systematic troubleshooting approach:

Sample Preparation Issues:

• Verify protein extraction efficiency with Bradford/BCA assays

• Ensure adequate protein loading (20-50 μg total protein)

• Test alternative extraction buffers optimized for nuclear proteins

• Include protease inhibitor cocktails to prevent degradation

Antibody-Related Factors:

• Verify antibody viability (avoid expired antibody)

• Increase antibody concentration (try 1:250 - 1:500 dilutions)

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

• Test different antibody lots if available

Detection System Adjustments:

• Use high-sensitivity ECL substrates for chemiluminescence

• Extend film exposure time or increase imaging duration

• Try signal enhancement systems (biotin-streptavidin amplification)

• Consider switching to fluorescent secondary antibodies for greater linearity

Biological Considerations:

• Confirm developmental stage/tissue known to express AGL14

• Consider environmental conditions that might affect expression

• Verify if your plant genotype has altered AGL14 expression

If the protein remains undetectable despite optimization, consider enrichment steps such as immunoprecipitation before Western blotting or using overexpression systems as positive controls.

What strategies can resolve high background or non-specific binding when using AGL14 Antibody?

High background with AGL14 Antibody can significantly impact data quality. Address this issue with these targeted approaches:

Blocking Optimization:

• Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

• Extend blocking time (2-3 hours at room temperature)

• Include 0.1-0.3% Tween-20 in washing and antibody dilution buffers

Washing Improvements:

• Increase washing duration (5-10 minutes per wash)

• Add additional washing steps (5-6 washes)

• Use higher salt concentration in wash buffers (up to 500 mM NaCl)

Antibody Adjustments:

• Further dilute primary antibody (1:1000 - 1:5000)

• Pre-absorb antibody with plant extract from non-expressing tissue

• Reduce secondary antibody concentration

• Use highly cross-adsorbed secondary antibodies

Membrane Handling:

• Minimize membrane drying during procedure

• Consider using PVDF instead of nitrocellulose (or vice versa)

• Test freshly prepared buffers

Technical Considerations:

• Ensure complete protein transfer to membrane

• Verify blocking agent compatibility with detection system

• Check for contamination in reagents

Document successful optimization conditions for future experiments to ensure reproducibility.

How can AGL14 Antibody be utilized in spatial expression studies of plant development?

AGL14 Antibody can be adapted for spatial expression analysis through these methodological approaches:

Immunohistochemistry (IHC) Protocol Development:

• Optimize tissue fixation (4% paraformaldehyde or 3:1 ethanol:acetic acid)

• Test different antigen retrieval methods (citrate buffer, pH 6.0)

• Use decreasing ethanol series for rehydration

• Block with 3-5% BSA or normal serum in PBS with 0.1% Triton X-100

• Apply primary antibody (1:100 - 1:500) overnight at 4°C

• Detect with fluorescent or enzyme-conjugated secondary antibodies

Whole-Mount Immunofluorescence:

• Modify sample clearing protocols (ClearSee or modified Pseudo-Schiff reagents)

• Extend antibody incubation times (24-48 hours)

• Use confocal microscopy for 3D visualization

Correlative Approaches:

• Combine with in situ hybridization for mRNA-protein correlation

• Use with cell-type specific markers to identify precise expression domains

• Implement with EdU labeling to correlate with cell division patterns

These approaches can provide valuable insights into AGL14 protein localization during key developmental processes in Arabidopsis thaliana, particularly in root and floral development where MADS-box transcription factors play critical roles.

What considerations should guide co-immunoprecipitation experiments using AGL14 Antibody?

Co-immunoprecipitation (Co-IP) with AGL14 Antibody can reveal protein interaction networks but requires careful optimization:

Extraction Buffer Composition:

• Include mild detergents (0.5-1% NP-40 or Triton X-100)

• Add protease and phosphatase inhibitors

• Test varying salt concentrations (100-300 mM NaCl)

• Include stabilizing agents (5-10% glycerol)

Protocol Optimization:

• Pre-clear lysates with Protein A/G beads to reduce background

• Test different antibody-to-lysate ratios (2-5 μg antibody per mg protein)

• Optimize incubation time (2 hours to overnight at 4°C)

• Use gentle washing conditions to preserve weak interactions

Controls:

• Input control (5-10% of starting material)

• IgG control (rabbit IgG at equivalent concentration)

• Reverse Co-IP with antibodies against suspected interacting partners

• Negative control using tissue without AGL14 expression

Validation Methods:

• Mass spectrometry identification of co-immunoprecipitated proteins

• Western blot verification of known or suspected interaction partners

• Functional validation through genetic studies

Since AGL14 is a transcription factor, expect interactions with other MADS-box proteins, chromatin modifiers, and transcriptional co-regulators. Consider crosslinking approaches for capturing transient nuclear interactions.

How can AGL14 Antibody be integrated with proteomics approaches for comprehensive protein interaction studies?

Integrating AGL14 Antibody with proteomics creates powerful research opportunities:

Sample Preparation Strategies:

• Use antibody-based enrichment prior to mass spectrometry

• Implement BioID or TurboID proximity labeling with AGL14 fusion proteins

• Compare results with conventional Co-IP to identify stable vs. transient interactors

Analysis Workflow:

• Perform immunoprecipitation with AGL14 Antibody

• Process samples for LC-MS/MS analysis

• Use label-free quantification or isotope labeling for comparative studies

• Apply appropriate statistical filtering (fold-change ≥2, p-value ≤0.05)

• Validate top candidates with reciprocal Co-IP or in vivo techniques

Data Integration Framework:

This integrative approach can reveal functional protein modules involved in AGL14-mediated developmental processes, providing insight beyond simple binary interactions.

What methodological considerations should guide cross-species application of AGL14 Antibody?

When applying AGL14 Antibody across different plant species, consider these methodological approaches:

Sequence Homology Assessment:

• Perform alignment of AGL14 orthologs across target species

• Identify conservation in the immunogenic region used to generate the antibody

• Calculate percent identity and similarity in epitope regions

Validation Strategy for Cross-Species Application:

• Begin with Western blot to confirm band at expected molecular weight

• Include positive control (Arabidopsis thaliana extract)

• Perform peptide competition assays with heterologous proteins

• Validate with genetic approaches where possible (knockdowns in target species)

Optimization for Different Species:

• Test increased antibody concentration for distantly related species

• Modify extraction buffers based on tissue-specific compounds

• Adjust incubation conditions (time, temperature)

• Consider species-specific blockers to reduce background

Expected Cross-Reactivity:

• High probability: Brassicaceae family members

• Moderate probability: Other eudicots with conserved MADS-box domains

• Lower probability: Monocots and gymnosperms

Document both successful and unsuccessful cross-species applications to contribute to the broader understanding of antibody specificity across plant lineages.