At4g20800 Antibody

What is At4g20800 and why is it significant for plant research?

At4g20800 is a gene encoding a FAD-binding Berberine family protein in Arabidopsis thaliana (mouse-ear cress), a model organism widely used in plant molecular biology. This protein functions in oxidoreductase activity, FAD binding, and catalytic activity, and is located primarily in the endomembrane system . Its significance stems from its expression patterns, particularly in embryo and synergid cells during specific developmental stages (C globular stage), suggesting important roles in reproductive development and female gametophyte function . Investigating this protein can provide valuable insights into plant reproduction, oxidative processes, and cellular signaling pathways critical for plant development.

What are the key structural and biochemical properties of the At4g20800 protein?

The At4g20800 protein exhibits several distinctive characteristics that influence antibody development and experimental applications:

• Molecular Weight: 58,849.90 Da

• Isoelectric Point (IEP): 9.44 (indicating a basic protein)

• GRAVY value: -0.16 (slightly hydrophilic)

• Length: 528 amino acids

• Key domains: Contains FAD-binding type 2 domain (InterPro:IPR016166), Berberine/berberine-like domain (InterPro:IPR012951), and FAD linked oxidase N-terminal domain (InterPro:IPR006094)

The protein sequence begins with "MKEVVYVLLLVLLVSVSDAN" and contains multiple functional regions that contribute to its oxidoreductase activity . Its relatively basic nature and specific domain architecture must be considered when developing experimental protocols using antibodies against this protein.

How should researchers select between polyclonal and monoclonal antibodies for At4g20800 research?

When investigating At4g20800, the choice between polyclonal and monoclonal antibodies depends on experimental objectives:

Polyclonal antibodies offer advantages for detecting At4g20800 in its native state as they recognize multiple epitopes, enhancing detection sensitivity and maintaining reactivity even if some epitopes are altered during experimental procedures. This makes them particularly useful for initial characterization studies, immunoprecipitation, and applications where protein conformation may vary.

Monoclonal antibodies provide superior specificity by targeting a single epitope, making them ideal for discriminating between At4g20800 and closely related FAD-binding proteins in the Arabidopsis genome. They ensure consistent results across experiments and are preferable for quantitative analyses requiring precise standardization.

For researchers studying developmental expression patterns in synergid cells or embryos, polyclonal antibodies might initially provide better detection in immunohistochemistry, while monoclonal antibodies would be valuable for distinguishing At4g20800 from related proteins in the Berberine family.

What sample preparation techniques are recommended for At4g20800 detection in plant tissues?

Effective sample preparation is crucial for successful At4g20800 detection due to its endomembrane localization and expression in specific cell types:

For protein extraction, researchers should:

• Use fresh tissue whenever possible, particularly when targeting embryos or reproductive structures where At4g20800 is expressed

• Employ a buffer system containing non-ionic detergents (0.5-1% Triton X-100 or NP-40) to solubilize membrane-associated proteins

• Include protease inhibitors to prevent degradation during extraction

• Consider subcellular fractionation techniques to enrich endomembrane fractions where At4g20800 is predominantly located

For immunohistochemistry in female gametophyte tissues:

• Fixation with 4% paraformaldehyde is recommended for preserving protein epitopes

• Consider specialized clearing protocols for reproductive tissues similar to those used in transcriptome analysis of ovules

• When working with ovules, collect approximately 2,500-3,000 samples from multiple plants to minimize plant-to-plant variations, following approaches used in transcriptome studies

How can researchers validate the specificity of At4g20800 antibodies?

Validating antibody specificity is essential for reliable research outcomes with At4g20800:

• Genetic controls: The gold standard validation employs At4g20800 knockout/knockdown lines compared with wild-type Arabidopsis. Complete absence or reduction of signal in mutant lines provides strong evidence for antibody specificity.

• Recombinant protein controls: Express and purify recombinant At4g20800 protein for Western blot validation. The antibody should detect a band at approximately 58.85 kDa corresponding to the predicted molecular weight .

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptide derived from At4g20800 sequence. Signal reduction or elimination confirms epitope-specific binding.

• Cross-reactivity assessment: Test for cross-reactivity with the closest Arabidopsis homolog (AT1G26420.1), which represents the best protein match according to TAIR10 annotations . This control is particularly important for ensuring the antibody specifically detects At4g20800.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry analysis to confirm the antibody captures At4g20800 rather than related FAD-binding proteins.

• Tissue-specific expression verification: Confirm detection patterns match known expression in embryo and synergid cells , with appropriate negative controls from tissues where expression is minimal.

What are the optimal immunohistochemistry protocols for localizing At4g20800 in female reproductive tissues?

Given At4g20800's expression in synergid cells and embryos, specialized immunohistochemistry protocols are required:

• Tissue preparation:

  • Collect ovules at late 12 to 13 floral stages for mature embryo sac analysis (FG5-FG7)

  • Fix tissues in 4% paraformaldehyde in PBS for 2-4 hours at 4°C

  • Perform clearing using chloral hydrate-based solution or methyl salicylate for better visualization of embryo sacs

  • Consider embedding in polyethylene glycol rather than paraffin to better preserve antigenic sites

• Antigen retrieval:

  • Perform citrate buffer (pH 6.0) heat-induced epitope retrieval to improve antibody access

  • For membrane-associated proteins like At4g20800, light protease treatment may enhance accessibility

• Detection system:

  • Use TSA (Tyramide Signal Amplification) for enhanced sensitivity when detecting low-abundance proteins

  • Counterstain with DAPI to visualize nuclei for precise localization within the embryo sac

  • Consider dual immunolabeling with cell-type markers for synergid cells to confirm cell-specific localization

• Controls:

  • Include wild-type vs. mutant tissue comparisons

  • Perform parallel experiments with pre-immune serum

  • Use tissues from developmental stages when expression is expected to be absent

• Imaging considerations:

  • Employ confocal microscopy for precise subcellular localization within the endomembrane system

  • Z-stack imaging is recommended for three-dimensional reconstruction of expression patterns in embryo sacs

What Western blot optimization strategies are recommended for At4g20800 detection?

Optimizing Western blot protocols for At4g20800 requires addressing several technical considerations:

• Sample preparation:

  • Enrich membrane fractions using ultracentrifugation to concentrate the endomembrane-localized At4g20800

  • Consider adding 6M urea to sample buffer for improved solubilization of membrane proteins

  • Use fresh tissue extracts whenever possible, as the FAD-binding domains may be sensitive to oxidation during storage

• Gel selection and transfer parameters:

  • Use 10% SDS-PAGE gels for optimal resolution around the expected 58.85 kDa size

  • For membrane proteins, semi-dry transfer with 20% methanol transfer buffer may yield better results

  • Consider longer transfer times (overnight at low voltage) to ensure complete transfer of the protein

• Blocking and antibody conditions:

  • Test both BSA and milk-based blocking solutions; the basic nature of At4g20800 (pI 9.44) may cause non-specific interactions with certain blocking agents

  • Optimize primary antibody dilutions (typically starting at 1:1000)

  • Extended incubation times at 4°C often improve specific signal for plant proteins

• Signal detection optimization:

  • For reproductive tissues with potentially low expression, consider chemiluminescent substrates with extended signal duration

  • Prepare dilution series of positive control samples to establish the detection limit

  • Use pre-stained markers that accurately indicate the expected molecular weight range

• Troubleshooting guidance:

  • If multiple bands appear, consider tissue-specific post-translational modifications

  • For weak signals, validate expression levels by referencing transcriptome data from similar tissues

  • For background issues, increase washing stringency with higher salt concentrations or detergent levels

How can At4g20800 antibodies be effectively used in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) experiments with At4g20800 require specific considerations due to its membrane association and biochemical properties:

• Buffer optimization:

  • Use buffers containing 0.5-1% NP-40 or digitonin to solubilize membrane proteins while preserving protein-protein interactions

  • Include 150-300 mM NaCl to reduce non-specific interactions, with optimization required for specific experimental conditions

  • Add protease inhibitors, reducing agents, and phosphatase inhibitors to preserve native interactions

• Crosslinking considerations:

  • For transient interactions, consider reversible crosslinkers like DSP (dithiobis(succinimidyl propionate))

  • Formaldehyde crosslinking (0.1-0.3%) may help preserve membrane protein interactions in their native environment

  • Optimize crosslinking time to balance between capturing interactions and maintaining antibody epitope accessibility

• Control experiments:

  • Perform parallel IPs with pre-immune serum or IgG from the same species

  • Include RNase treatment controls if RNA-mediated interactions are suspected

  • Consider isotype-matched control antibodies for monoclonal antibody Co-IPs

• Elution and analysis strategies:

  • Test both harsh elution (with SDS and heat) and native elution (with competing peptides)

  • For mass spectrometry analysis, consider on-bead digestion to minimize contamination

  • When analyzing results, compare with known interactors of proteins containing similar domains (FAD-binding, Berberine-like) to identify potential conserved interaction networks

• Verification approaches:

  • Confirm key interactions with reverse Co-IP experiments

  • Validate physiologically relevant interactions using bimolecular fluorescence complementation or FRET in plant cells

  • Cross-reference with data from SUBAcon plant protein interaction databases

What approaches are recommended for studying At4g20800 expression patterns across developmental stages?

To comprehensively analyze At4g20800 expression patterns:

• Temporal sampling strategy:

  • For reproductive tissues, collect ovules at defined floral stages (12-13) as established in transcriptome studies

  • Sample multiple developmental time points, particularly focusing on the C globular stage where expression has been documented

  • Consider stratified sampling of different organs to establish a comprehensive expression map

• Comparative analysis framework:

  • Use both antibody-based detection (Western blot, immunohistochemistry) and transcript analysis (qRT-PCR)

  • For quantitative comparisons, normalize protein levels to established housekeeping proteins

  • Reference existing transcriptome datasets that include At4g20800 expression data in various tissues

• Single-cell resolution techniques:

  • For female gametophyte studies, employ laser capture microdissection followed by protein extraction

  • Consider reporter gene fusions (GUS or fluorescent proteins) to complement antibody-based approaches

  • Immunogold electron microscopy can provide subcellular resolution within specific cell types

• Data validation and integration:

  • Compare protein expression patterns with transcriptome data

  • Consider analyzing multiple accessions/ecotypes to identify potential expression variations

  • Integrate expression data with functional studies to correlate expression patterns with developmental phenotypes

• Experimental design considerations:

  • Include biological replicates from independent plants (minimum 3) to account for plant-to-plant variation

  • For reproductive tissue studies, pool material from multiple plants (2,500-3,000 ovules) as practiced in transcriptome studies

  • Document growth conditions meticulously as environmental factors may influence expression patterns

How should researchers address non-specific binding issues with At4g20800 antibodies?

Non-specific binding can significantly impact experimental outcomes when working with At4g20800 antibodies:

• Diagnostic indicators of non-specificity:

  • Multiple unexpected bands on Western blots

  • Signal in tissues where At4g20800 is not expressed based on transcriptome data

  • Persistent signal in knockout/knockdown lines

  • Inconsistent localization patterns in immunohistochemistry

• Optimization strategies:

  • Increase antibody dilution systematically (1:500, 1:1000, 1:2000) to identify optimal signal-to-noise ratio

  • Test alternative blocking agents (5% BSA, 5% milk, commercial blockers) to identify optimal formulation

  • Add competing proteins from non-plant sources to reduce non-specific interactions

  • Increase washing stringency with higher salt concentrations (150mM to 500mM NaCl) or detergent levels

• Purification approaches:

  • Consider affinity purification of antibodies against recombinant At4g20800 protein

  • For polyclonal antibodies, pre-absorb with Arabidopsis extracts from knockout lines to remove antibodies recognizing non-target proteins

  • For critical applications, consider immunodepletion against closely related proteins, particularly AT1G26420.1

• Application-specific optimization:

  • For Western blots, extend blocking time and add 0.1-0.5% Tween-20 to antibody dilution buffers

  • For immunohistochemistry, include 1-5% normal serum from the secondary antibody host species

  • For IP/Co-IP experiments, use more stringent wash buffers containing up to 0.1% SDS

What strategies can resolve weak or absent signals when detecting At4g20800?

When faced with weak or undetectable At4g20800 signals:

• Sample preparation optimization:

  • Verify tissue selection matches known expression patterns (embryo, synergid)

  • Increase starting material quantity, especially for reproductive tissues

  • Consider subcellular fractionation to concentrate endomembrane components where At4g20800 is located

  • Add protease inhibitor cocktails to prevent degradation during extraction

• Protocol enhancement techniques:

  • For Western blots, increase protein loading (50-100 μg per lane)

  • Employ signal amplification systems (TSA for immunohistochemistry, enhanced chemiluminescence for Western blots)

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

  • Consider alternative detergents for extraction (CHAPS, Brij-35) if standard NP-40 or Triton X-100 proves insufficient

• Technical optimization approaches:

  • Test alternative fixation methods for immunohistochemistry

  • For proteins with basic pI like At4g20800 (pI 9.44), adjust transfer buffer pH slightly

  • Consider using PVDF rather than nitrocellulose membranes for Western blots of basic proteins

  • Verify secondary antibody compatibility and activity with simple dot blot tests

• Validation approaches when signal remains problematic:

  • Confirm At4g20800 expression in your specific samples via RT-PCR

  • Test antibody reactivity against recombinant At4g20800 protein

  • Consider epitope exposure issues and test antigen retrieval methods

How can At4g20800 antibodies be used to investigate protein-protein interactions in FAD-binding protein complexes?

Investigating protein-protein interactions involving At4g20800 requires specialized approaches:

• Proximity-based interaction methods:

  • BioID or TurboID fusion proteins can identify neighboring proteins in plant cells without requiring stable interactions

  • Split-ubiquitin yeast two-hybrid systems are suitable for membrane-associated proteins like At4g20800

  • FRET-FLIM (Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging) can detect direct interactions in planta

• Co-immunoprecipitation optimization for membrane proteins:

  • Use mild detergents (0.5% digitonin) to preserve membrane protein complexes

  • Consider formaldehyde crosslinking (0.1-0.3%) prior to cell lysis

  • Employ two-step purification strategies (tandem affinity purification) for higher stringency

  • Scale up starting material (up to 5-10g tissue) to compensate for potential low abundance

• Domain-specific interaction mapping:

  • Create domain-specific antibodies targeting the FAD-binding domain (InterPro:IPR016166), Berberine-like domain (InterPro:IPR012951), and FAD-linked oxidase domain (InterPro:IPR006094)

  • Use these domain-specific antibodies to determine which regions mediate specific interactions

  • Consider competitive peptide approaches to disrupt specific domain interactions

• Dynamic interaction analysis:

  • Study interaction changes during developmental progression, particularly during female gametophyte development

  • Investigate stress-induced interaction modifications by exposing plants to oxidative stress before analysis

  • Compare interaction profiles between different tissues where At4g20800 is expressed (embryo versus synergid cells)

What are the considerations for developing phospho-specific antibodies targeting At4g20800?

Researchers interested in At4g20800 phosphorylation should consider:

• Phosphorylation site prediction and selection:

  • Use phosphoproteomics databases and prediction algorithms to identify potential phosphorylation sites

  • Prioritize conserved residues across related FAD-binding proteins

  • Consider sites within functional domains that might regulate activity

  • Select sites with favorable surrounding sequences for antibody generation (adequate hydrophilicity and antigenicity)

• Phospho-specific antibody production strategies:

  • Generate antibodies against synthetic phosphopeptides containing the phosphorylated residue of interest

  • Consider dual purification: positive selection with phosphopeptide followed by negative selection against non-phosphorylated peptide

  • For multiple phosphorylation sites, develop individual antibodies for each site rather than attempting to create antibodies recognizing multiple phosphorylations

• Validation requirements:

  • Test antibody specificity using phosphatase-treated samples as negative controls

  • Verify against recombinant proteins with site-directed mutagenesis (phospho-mimetic and non-phosphorylatable versions)

  • Confirm detection of endogenous phosphorylated protein induced by relevant stimuli

  • Include phosphopeptide competition assays to confirm epitope specificity

• Experimental applications:

  • Investigate phosphorylation status changes during development or in response to stress

  • Use phospho-specific antibodies to purify and identify interaction partners specific to the phosphorylated form

  • Consider quantitative applications such as ELISA to measure phosphorylation levels across conditions

How can At4g20800 antibodies contribute to understanding the role of this protein in female gametophyte development?

At4g20800's expression in synergid cells suggests important roles in female gametophyte development:

• Developmental timing studies:

  • Use stage-specific immunolocalization to track At4g20800 expression throughout female gametophyte development

  • Compare expression patterns in wild-type versus mutant ovules with developmental defects

  • Correlate protein levels with functional stages of synergid development and function

• Functional analysis approaches:

  • Employ antibody microinjection to temporarily inhibit At4g20800 function in developing ovules

  • Use immunoprecipitation followed by activity assays to determine if developmental regulation occurs through protein modification rather than expression changes

  • Combine with genetic approaches (RNAi, CRISPR) to correlate protein elimination with developmental phenotypes

• Cell-type specific analysis:

  • Perform co-localization studies with known synergid markers

  • Investigate potential polarized distribution within synergid cells using high-resolution immunolocalization

  • Compare expression between synergids and other female gametophyte cells to determine exclusivity

• Comparative approaches:

  • Analyze At4g20800 expression across Arabidopsis ecotypes to identify natural variation

  • Extend studies to related species to determine evolutionary conservation of expression patterns

  • Correlate expression with morphological or functional differences in female gametophyte development

• Integration with transcriptome data:

  • Compare protein expression patterns with transcriptome data from female gametophyte studies

  • Investigate potential post-transcriptional regulation by comparing mRNA and protein levels

  • Use existing transcriptome comparisons between wild-type and developmental mutants to identify potential regulatory networks

How should researchers approach quantitative analysis of At4g20800 expression data?

Quantitative analysis of At4g20800 expression requires rigorous approaches:

• Normalization strategies for Western blot analysis:

  • Use multiple loading controls appropriate for plant tissues (actin, tubulin, GAPDH)

  • Consider specialized loading controls for membrane fraction analysis

  • Employ total protein normalization methods (Ponceau S, SYPRO Ruby) to avoid issues with variable reference protein expression

  • Validate antibody linearity by analyzing dilution series of positive control samples

• Image analysis optimization:

  • Use software with background subtraction capabilities (ImageJ with appropriate plugins)

  • Establish clear rules for defining region of interest boundaries

  • Consider fluorescent Western blotting for wider linear dynamic range compared to chemiluminescence

  • Apply consistent analysis parameters across all experimental replicates

• Statistical approaches:

  • Use appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Account for biological variability by analyzing samples from multiple independent plants

  • Consider power analysis to determine appropriate sample sizes for detecting biologically relevant changes

  • Report effect sizes along with p-values to indicate biological significance

• Integrated multi-method quantification:

  • Correlate protein levels (antibody-based detection) with transcript levels (qRT-PCR)

  • Consider absolute quantification using purified recombinant protein standards

  • Compare different detection methods (Western blot vs. ELISA) to validate quantitative trends

What data interpretation challenges are specific to At4g20800 research?

Researchers should be aware of specific interpretation challenges:

• Expression context considerations:

  • Low abundance in whole-tissue extracts due to cell-type specific expression (synergid, embryo)

  • Potential developmental or environmental regulation affecting detection

  • Possible post-translational modifications altering antibody recognition

  • Membrane association potentially affecting extraction efficiency

• Comparative analysis frameworks:

  • When comparing mutant and wild-type samples, consider secondary effects of mutation on tissue composition

  • For developmental studies, ensure precise staging to avoid misinterpreting temporal changes

  • When analyzing protein-protein interactions, consider that membrane protein interactions may be more sensitive to experimental conditions

• Addressing conflicting results:

  • Reconcile differences between protein and transcript levels by considering post-transcriptional regulation

  • When immunolocalization and biochemical fractionation show discrepancies, consider fixation or extraction artifacts

  • For inconsistencies between antibody-based detection and reporter gene approaches, evaluate the limitations of each method

• Integration with existing knowledge:

  • Compare At4g20800 findings with data on related FAD-binding proteins

  • Consider the biological implications of expression in synergid cells in the context of known synergid functions

  • Interpret novel interaction partners in the context of known berberine family protein functions