At4g26220 Antibody

What is At4g26220 and what function does it serve in Arabidopsis thaliana?

At4g26220 is a gene that encodes a caffeoyl-coenzyme A O-methyltransferase (CCoAOMT)-like protein in Arabidopsis thaliana. It belongs to the S-adenosyl-L-methionine-dependent methyltransferases superfamily of proteins. This enzyme has a distinctive catalytic profile with a strong preference for methylating the para position of flavanones and dihydroflavonols, while it methylates flavones and flavonols in the meta-position . The protein is also known by the alias CCoAOMT7 in scientific literature . The molecular function of At4g26220 has been experimentally demonstrated to involve caffeoyl-CoA O-methyltransferase activity . In terms of cellular localization, the At4g26220 protein has been confirmed to be present in the cytosol through direct assay (IDA) and transcript analysis (TAS) .

What are the primary applications for At4g26220 antibodies in plant research?

At4g26220 antibodies have demonstrated utility in several key applications in plant research. Based on validated experimental protocols, these antibodies are particularly suitable for Western blot (WB) analysis and Enzyme-Linked Immunosorbent Assay (ELISA) . When designing experiments with At4g26220 antibodies, researchers should consider the antibody's species reactivity, which has been specifically tested and confirmed for plants, particularly Arabidopsis thaliana . The availability of purified antibodies, often through antigen affinity methods, makes them suitable for detecting native protein levels in plant tissues. Additionally, having access to both antigens (for positive controls) and pre-immune serum (for negative controls) enables proper experimental validation and troubleshooting in research settings .

What considerations are important when selecting an At4g26220 antibody for experimental use?

When selecting an At4g26220 antibody for experimental use, researchers should consider several critical factors:

• Antibody type and host species: Currently available At4g26220 antibodies include rabbit polyclonal antibodies . The host species affects cross-reactivity and compatibility with secondary detection systems.

• Validation status: Review whether the antibody has been validated for specific applications such as Western blot, ELISA, or immunofluorescence. For At4g26220, commercially available antibodies have been validated for ELISA and Western blot applications .

• Immunogen information: Understanding the immunogen used to generate the antibody is critical. For At4g26220, recombinant Arabidopsis thaliana At4g26220 protein has been used as the immunogen , which may influence epitope recognition.

• Purification method: Antibodies purified by antigen affinity methods typically offer higher specificity. At4g26220 antibodies available commercially are purified using antigen affinity techniques .

• Storage conditions: Proper storage (-20°C or -80°C) is essential to maintain antibody integrity and performance over time .

What is the recommended protocol for using At4g26220 antibodies in Western blot analysis?

When using At4g26220 antibodies for Western blot analysis, the following optimized protocol is recommended:

Sample Preparation:

• Extract total proteins from plant tissue using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Quantify protein concentration using Bradford or BCA assay.

• Mix 20-50μg of protein with Laemmli buffer and heat at 95°C for 5 minutes.

SDS-PAGE and Transfer:

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

• Transfer proteins to PVDF or nitrocellulose membrane at 100V for 60-90 minutes.

Antibody Incubation:

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

• Incubate with rabbit polyclonal At4g26220 antibody (dilution 1:1000 to 1:5000 in TBST with 1% BSA) overnight at 4°C .

• Wash three times with TBST, 5 minutes each.

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1 hour at room temperature.

• Wash three times with TBST, 5 minutes each.

Detection:

• Apply ECL substrate and image using a chemiluminescence imaging system.

• Expected molecular weight of At4g26220 protein is approximately 27-30 kDa.

Validation Controls:

• Use the provided 200μg antigen as a positive control .

• Include pre-immune serum (provided with commercial antibodies) as a negative control .

How can researchers optimize At4g26220 antibody performance in ELISA?

To optimize At4g26220 antibody performance in ELISA, researchers should implement this protocol with particular attention to critical optimization steps:

Indirect ELISA Protocol:

• Coat 96-well plates with plant protein extract or purified recombinant At4g26220 (0.1-1μg/well) in coating buffer (50mM carbonate-bicarbonate, pH 9.6) overnight at 4°C.

• Wash 3 times with PBST (PBS + 0.05% Tween-20).

• Block with 1-5% BSA or 5% non-fat milk in PBST for 1-2 hours at room temperature.

• Add At4g26220 antibody at various dilutions (starting from 1:500 to 1:5000) in blocking buffer and incubate for 2 hours at room temperature or overnight at 4°C .

• Wash 4 times with PBST.

• Add HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000-1:10000 dilution and incubate for 1 hour.

• Wash 4 times with PBST.

• Add substrate (TMB) and measure absorbance at 450nm after stopping the reaction with 2N H₂SO₄.

Critical Optimization Points:

• Antibody titration: Test several dilutions of primary antibody to determine optimal signal-to-noise ratio.

• Blocking agent: Compare BSA vs. non-fat milk to minimize background.

• Incubation temperature and time: Compare room temperature (1-2 hours) vs. 4°C (overnight) incubation.

• Controls: Include the provided positive control antigen and negative control pre-immune serum in every experiment .

• Sample preparation: Test different extraction buffers to maximize target protein solubility while preserving epitope recognition.

How can At4g26220 antibodies be used to investigate methyltransferase activity in plant tissues?

At4g26220 antibodies can be powerful tools for investigating methyltransferase activity through these methodological approaches:

Immunoprecipitation-Coupled Enzyme Activity Assay:

• Prepare plant tissue extracts in a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 0.1% NP-40, 5mM EDTA, and protease inhibitor cocktail.

• Incubate 500-1000μg of protein extract with 2-5μg of At4g26220 antibody overnight at 4°C with gentle rotation .

• Add Protein A/G magnetic beads and incubate for 2-4 hours at 4°C.

• Wash beads 4 times with washing buffer.

• Resuspend beads in methyltransferase assay buffer containing S-adenosyl-L-methionine as methyl donor and appropriate substrate (flavanones or dihydroflavonols) .

• Incubate at 30°C for 30-60 minutes.

• Analyze methylated products by HPLC or LC-MS.

Tissue-Specific Expression Analysis:

• Extract proteins from different plant tissues (roots, stems, leaves, flowers).

• Perform Western blot using At4g26220 antibody following the protocol above .

• Quantify relative protein levels across tissues.

• Correlate protein expression with methyltransferase activity by conducting parallel enzymatic assays.

Subcellular Localization Studies:

• Prepare subcellular fractions (cytosol, microsomes, chloroplasts).

• Confirm fractionation quality using marker antibodies for different compartments.

• Detect At4g26220 using the specific antibody via Western blot .

• Correlate localization with the known cytosolic localization from GO annotation .

What approaches can resolve contradictory findings when using At4g26220 antibodies across different experimental systems?

When facing contradictory results with At4g26220 antibodies across different experimental systems, researchers should implement these systematic troubleshooting approaches:

Antibody Validation Strategy:

• Epitope mapping: Determine the exact epitope recognized by the antibody and assess whether post-translational modifications might affect recognition.

• Knockout/knockdown controls: Use CRISPR/Cas9-edited Arabidopsis lines or RNAi knockdown plants lacking At4g26220 expression as negative controls.

• Cross-reactivity assessment: Test the antibody against recombinant proteins from related methyltransferase family members to assess specificity.

Experimental Condition Standardization:

• Create a detailed comparison table of differing parameters between experimental systems:

• Systematically test each parameter to identify the source of discrepancy.

Technical Adaptations:

• Epitope retrieval methods: If working with fixed tissues, compare different antigen retrieval methods.

• Signal amplification: For low abundance detection, implement tyramide signal amplification or other amplification methods.

• Alternative antibody formats: If possible, test monoclonal vs. polyclonal antibodies against different regions of the protein.

How does At4g26220 (CCoAOMT7) research relate to broader studies of plant methyltransferases?

At4g26220 (CCoAOMT7) research provides critical insights into the wider family of plant methyltransferases through several significant connections:

Evolutionary Context:
The CCoAOMT family in plants represents an important group of O-methyltransferases that participate in various metabolic pathways. At4g26220 specifically belongs to the caffeoyl-CoA O-methyltransferase subfamily, which plays roles in lignin biosynthesis and specialized metabolite production. Understanding the substrate specificity of At4g26220, which preferentially methylates the para position of flavanones and dihydroflavonols and the meta-position of flavones and flavonols , provides comparative data to other methyltransferases with different preferences.

Metabolic Network Integration:
At4g26220's function intersects with several important metabolic pathways:

• Phenylpropanoid pathway regulation

• Flavonoid biosynthesis and modification

• Potential roles in plant defense compound production

By studying At4g26220 with specific antibodies, researchers can illuminate how different methyltransferases coordinate their activities within these interconnected pathways.

Structural-Functional Relationships:
As a member of the S-adenosyl-L-methionine-dependent methyltransferases superfamily , At4g26220 shares structural features with numerous other methyltransferases. Antibody-based studies that capture protein-protein interactions or conformational states can reveal how structural differences translate to functional specialization among methyltransferase family members.

What emerging research directions could benefit from advances in At4g26220 antibody methodologies?

Several cutting-edge research areas stand to benefit significantly from improved At4g26220 antibody methodologies:

Synthetic Biology Applications:
Engineered methyltransferases based on At4g26220 could be used to create novel plant metabolites with pharmaceutical or industrial applications. Advanced antibody methods like conformational-specific antibodies could help characterize protein engineering success by detecting properly folded variants.

Climate Change Adaptation Research:
Methyltransferases may play roles in plant adaptation to changing environmental conditions. At4g26220 antibodies could be used to:

• Analyze protein expression changes under drought, temperature stress, or elevated CO₂

• Identify post-translational modifications that regulate enzyme activity under stress

• Study protein turnover rates under varying conditions

Systems Biology Integration:
The integration of At4g26220 studies into larger systems biology approaches requires advanced antibody applications:

• Spatial proteomics: Using At4g26220 antibodies with multiplexed immunofluorescence to map protein localization at tissue and cellular levels

• Temporal dynamics: Employing antibodies in time-course studies to understand dynamic changes in protein levels during development or stress responses

• Interactome mapping: Utilizing At4g26220 antibodies for immunoprecipitation followed by mass spectrometry to identify interaction partners

Machine Learning Applications:
Recent advances in active learning approaches for antibody-antigen binding prediction could be applied to optimize At4g26220 antibody design or selection. These computational methods could potentially:

• Predict epitope accessibility under different experimental conditions

• Optimize antibody sequences for improved specificity and affinity

• Design multiplexed detection systems with minimal cross-reactivity

What strategies can overcome common technical challenges when working with At4g26220 antibodies?

Researchers working with At4g26220 antibodies frequently encounter several technical challenges. Here are methodological solutions for each:

Challenge: Low Signal Intensity
Solutions:

• Signal amplification: Implement tyramide signal amplification (TSA) for immunohistochemistry or chemiluminescent substrates with extended reaction times for Western blots.

• Sample enrichment: Use subcellular fractionation to concentrate cytosolic proteins where At4g26220 is localized .

• Antibody concentration optimization: Systematically test concentration ranges from 1:500 to 1:5000 to identify the optimal signal-to-noise ratio .

• Extended incubation: Increase primary antibody incubation time to overnight at 4°C rather than shorter room temperature incubations.

Challenge: Non-specific Binding
Solutions:

• Optimized blocking: Test different blocking agents (BSA, non-fat milk, commercial blocking buffers) at concentrations from 1-5%.

• Pre-adsorption strategy: Incubate antibody with proteins from knockout plants lacking At4g26220 to remove antibodies that bind to other plant proteins.

• Stringent washing: Increase TBST/PBST washing steps (number and duration) after antibody incubations.

• Pre-immune serum comparison: Always run parallel experiments with the provided pre-immune serum to identify non-specific signals .

Challenge: Inconsistent Results Across Experiments
Solutions:

• Standard operating procedure (SOP): Develop and strictly adhere to a detailed SOP covering all experimental steps.

• Positive control inclusion: Include the provided antigen as a positive control in every experiment .

• Antibody aliquoting: Upon receipt, aliquot antibodies to minimize freeze-thaw cycles and maintain consistency.

• Batch processing: Process all comparative samples in the same experimental batch to minimize technical variation.

Several innovative approaches are emerging that could significantly enhance At4g26220 antibody applications in plant research:

Single-Cell Antibody Applications:

• Single-cell proteomics approaches using At4g26220 antibodies could reveal cell-type specific expression patterns and functional roles not detectable in whole-tissue analysis.

• Microfluidic antibody-based detection systems may enable high-throughput screening of At4g26220 expression across diverse genetic backgrounds or environmental conditions.

Antibody Engineering Approaches:

• Recombinant antibody technology allows for the production of engineered antibody fragments (Fab, scFv) with potentially improved penetration into plant tissues.

• Site-specific conjugation of fluorophores or enzymes to At4g26220 antibodies could enhance detection sensitivity without compromising binding properties.

Computational Optimization:
The application of active learning algorithms, as described in recent literature on antibody-antigen binding , could significantly improve At4g26220 antibody design and selection. These approaches could reduce the required experimental dataset size by up to 35% while accelerating the learning process about optimal binding conditions .

How can researchers integrate At4g26220 antibody data with other -omics approaches?

The integration of At4g26220 antibody-generated data with other -omics approaches creates powerful multi-dimensional insights:

Integrative Analysis Framework:

• Proteomics-Transcriptomics Integration: Correlate At4g26220 protein levels (detected by antibodies) with mRNA expression data to identify post-transcriptional regulation mechanisms.

• Metabolomics Connection: Link At4g26220 protein abundance with levels of methylated flavonoids and other methylated metabolites to establish structure-function relationships.

• Phenomics Correlation: Associate At4g26220 expression patterns with phenotypic traits under various environmental conditions.

Multi-omics Data Integration Methods:

• Network Analysis: Position At4g26220 within protein-protein interaction networks using antibody-based co-immunoprecipitation data combined with transcriptome-derived co-expression networks.

• Temporal and Spatial Mapping: Create 4D maps of At4g26220 expression during development and stress responses by combining antibody-based imaging with transcriptomics data from various tissues and time points.

• Causal Modeling: Develop causal models that predict how changes in At4g26220 activity affect downstream pathways by integrating proteomic and metabolomic data.