At2g27360 Antibody

What is AT2G27360 and why is it relevant to plant research?

AT2G27360 encodes a GDSL-like Lipase/Acylhydrolase superfamily protein in Arabidopsis thaliana that functions in hydrolase activity, specifically acting on ester bonds with carboxylesterase activity. This protein is involved in glycerol biosynthetic processes and lipid metabolism, making it significant for studying plant lipid pathways. The protein is expressed in 20 plant structures across 13 growth stages and is primarily localized in the endomembrane system, with SUBAcon data indicating strong extracellular localization (score: 1.000). Understanding this protein contributes to our knowledge of plant lipid metabolism and potentially stress responses, as many GDSL-family proteins are involved in plant defense mechanisms .

What are the key biochemical properties of the AT2G27360 protein that influence antibody production?

The AT2G27360 protein has several properties that should be considered when developing antibodies: it has a molecular weight of 43856.20 Da, an isoelectric point (IEP) of 4.97, a GRAVY score of -0.12 (slightly hydrophilic), and consists of 394 amino acids. The protein contains the InterPro domain Lipase, GDSL (IPR001087). These properties affect epitope accessibility, with the slightly negative charge at physiological pH potentially influencing antibody binding. The protein's subcellular localization in the endomembrane system and potential extracellular presence must be considered when designing extraction protocols for antibody validation experiments .

What extraction methods are most effective for isolating AT2G27360 for antibody validation?

For optimal extraction of AT2G27360 protein from Arabidopsis tissues, specialized extraction buffers designed for total soluble/membrane proteins are recommended. Extraction buffers optimized for subsequent western blot detection in denatured conditions, such as those provided in commercial kits (like AS08 300), have proven effective. The protocol should include fine grinding of plant tissue in liquid nitrogen, followed by extraction in buffer containing appropriate detergents (typically 2-3% SDS), reducing agents, and protease inhibitors. Given the protein's endomembrane localization, a two-phase extraction protocol may be necessary to maximize yield, first extracting soluble proteins followed by membrane-associated proteins .

How should Western blot protocols be optimized for AT2G27360 detection?

For Western blot detection of AT2G27360, consider the following methodology:

• Sample preparation: Extract protein using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels to achieve optimal separation at the ~44kDa range.

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C on PVDF membranes.

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute anti-AT2G27360 antibody at 1:1000 to 1:2000 in 2% BSA/TBST and incubate overnight at 4°C.

• Secondary antibody: Incubate with HRP-conjugated secondary antibody at 1:25,000 dilution for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence system with exposure times ranging from 30 seconds to 5 minutes.
  This protocol should be validated first with positive controls and may require optimization based on specific antibody characteristics .

What challenges are commonly encountered when using immunoprecipitation with AT2G27360 antibodies?

Immunoprecipitation (IP) of AT2G27360 presents several methodological challenges:

• Low abundance: As a lipase family protein, AT2G27360 may be expressed at relatively low levels in certain tissues or developmental stages.

• Membrane association: Its endomembrane localization requires careful buffer selection to solubilize without denaturing epitopes.

• Cross-reactivity: The GDSL lipase family contains multiple homologous proteins in Arabidopsis (closest match being AT1G28610.2), potentially causing cross-reactivity.
  To address these challenges:

• Use a mild IP buffer (25mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA) with protease inhibitors

• Pre-clear lysates with protein A/G beads to reduce nonspecific binding

• Validate antibody specificity using knockout lines

• Consider crosslinking antibodies to beads to prevent heavy chain interference in downstream applications

• Perform sequential elution with increasing stringency to optimize specific protein recovery

How can AT2G27360 antibodies be utilized for co-immunoprecipitation to identify protein interaction networks?

When utilizing AT2G27360 antibodies for co-immunoprecipitation (co-IP) studies to map protein interaction networks, researchers should implement the following methodological approach:

• Crosslinking strategy: Employ in vivo crosslinking with 1-2% formaldehyde for 10 minutes to capture transient interactions, especially important for enzyme-substrate relationships common in lipases.

• Extraction buffer optimization: Use a two-phase extraction protocol with gentle detergents (0.5% digitonin or 1% NP-40) to maintain native protein complexes while effectively solubilizing membrane-associated proteins.

• IP validation controls: Include both technical controls (IgG control, input samples) and biological controls (AT2G27360 knockout lines) to distinguish genuine interactors from nonspecific binding.

• Sequential elution: Implement a gradient elution strategy starting with competitive elution using AT2G27360 peptides followed by more stringent conditions.

• Mass spectrometry analysis: Submit samples for LC-MS/MS analysis using both data-dependent and data-independent acquisition methods to maximize detection of low-abundance interactors.

• Interaction validation: Confirm key interactions through reciprocal co-IP and orthogonal methods such as yeast two-hybrid or bimolecular fluorescence complementation.
  This comprehensive approach helps establish the biological context of AT2G27360 within the plant lipid metabolism and signaling networks .

What strategies should be employed for antibody validation in AT2G27360 knockout or knockdown lines?

A rigorous validation strategy for AT2G27360 antibodies using genetic knockout/knockdown lines involves:

• Genetic material selection:

  • T-DNA insertion lines targeting AT2G27360 (available from stock centers)

  • CRISPR/Cas9-generated knockout lines

  • RNAi or amiRNA knockdown lines (for partial reduction)

• Validation protocol:

  • Extract protein from wild-type, heterozygous, and homozygous knockout tissues using identical conditions

  • Perform Western blot analysis with consistent loading (confirmed via loading controls like anti-actin)

  • Compare band intensity at expected molecular weight (~44 kDa)

  • Document complete absence of signal in knockouts or proportional reduction in knockdowns

• Secondary confirmation:

  • Perform RT-qPCR to confirm transcript absence/reduction

  • Include recombinant AT2G27360 protein as a positive control

  • Test for cross-reactivity with the closest homolog (AT1G28610.2)
    This systematic approach provides definitive evidence of antibody specificity and determines detection limits, critical for subsequent experimental applications .

How can immunolocalization techniques be optimized for studying AT2G27360 subcellular distribution?

For precise immunolocalization of AT2G27360, which is predicted to localize primarily to the endomembrane system with potential extracellular presence (SUBAcon score: 1.000), implement this methodology:

• Tissue preparation protocol:

  • Fix tissue samples in 4% paraformaldehyde for 2 hours at room temperature

  • Perform stepwise dehydration through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Embed in either paraffin for light microscopy or LR White resin for electron microscopy

  • Section at 5-10 μm thickness for light microscopy or 70-90 nm for electron microscopy

• Immunolabeling optimization:

  • Test multiple epitope retrieval methods (citrate buffer at pH 6.0, enzymatic treatment with proteinase K)

  • Block with 3% BSA, 0.1% Triton X-100 in PBS for 1 hour

  • Incubate with primary antibody at 1:100-1:500 dilutions overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies for confocal microscopy or gold-conjugated antibodies for EM

• Colocalization analysis:

  • Co-label with established endomembrane markers (e.g., anti-BiP for ER, anti-SYP61 for TGN)

  • Perform quantitative colocalization analysis using Pearson's or Manders' coefficients

  • Compare observed localization with predicted patterns from SUBA database
    This approach allows accurate determination of AT2G27360's dynamic localization across different tissue types and developmental stages .

How should researchers interpret conflicting results between antibody-based detection and transcript analysis of AT2G27360?

When facing discrepancies between AT2G27360 protein detection (via antibodies) and transcript analysis (via RT-PCR/RNA-seq), consider this analytical framework:

• Potential causes of discrepancy:

  • Post-transcriptional regulation: miRNA targeting, alternative splicing

  • Translational control: ribosome occupancy, upstream ORFs

  • Protein stability differences: half-life variations across conditions

  • Technical limitations: antibody sensitivity vs. transcript detection limits

• Resolution methodology:

  • Temporal analysis: Track both transcript and protein levels across multiple timepoints

  • Polysome profiling: Determine if transcripts are actively translated

  • Protein stability assays: Use cycloheximide chase experiments to measure turnover rates

  • Absolute quantification: Employ targeted proteomics (MRM/PRM) with synthetic peptide standards

• Data integration approach:

  • Integrate data using statistical methods that account for measurement error

  • Normalize expression values appropriately across techniques

  • Develop mathematical models that incorporate delay between transcription and translation
    This systematic analysis helps distinguish between technical artifacts and genuine biological phenomena, such as stress-induced post-translational modifications that might affect antibody recognition .

What statistical approaches are most appropriate for quantifying AT2G27360 protein levels across different experimental conditions?

For robust quantification of AT2G27360 protein levels across experimental conditions, implement this statistical framework:

• Experimental design considerations:

  • Minimum of 3-4 biological replicates per condition

  • Technical replicates (2-3) for immunoblotting

  • Inclusion of concentration gradients for standard curves

  • Randomization of sample processing order

• Quantification methodology:

  • Densitometry analysis of Western blots using standardized software (ImageJ/Fiji)

  • Normalization to multiple housekeeping proteins (actin, tubulin, GAPDH)

  • Use of loading controls to account for extraction efficiency variation

• Statistical analysis protocol:

  • Test for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

  • Apply appropriate transformations if data is non-normal (log, square root)

  • Perform ANOVA followed by post-hoc tests (Tukey's HSD) for multiple comparisons

  • Calculate effect sizes (Cohen's d) to quantify magnitude of differences

  • Implement linear mixed-effects models for complex experimental designs

• Visualization standards:

  • Present data with scatter plots overlaid on box plots or violin plots

  • Include error bars representing standard deviation or standard error

  • Indicate statistical significance using consistent notation
    This comprehensive approach minimizes variability and allows for reliable quantitative comparisons across different tissues, treatments, or genetic backgrounds .

What are the most common causes of non-specific binding with AT2G27360 antibodies and how can they be addressed?

Non-specific binding is a common challenge with plant protein antibodies, particularly for members of multi-gene families like GDSL lipases. Address these issues with the following methodology:

• Sources of non-specificity:

  • Cross-reactivity with homologous proteins (particularly AT1G28610.2)

  • Post-translational modifications altering epitope recognition

  • Incomplete blocking during immunoblotting

  • Sample preparation artifacts (protein aggregation, degradation)

• Optimization strategy:

  • Blocking optimization: Test different blocking agents (5% BSA vs. 5% milk) and durations

  • Antibody dilution: Perform titration experiments (1:500 to 1:5000) to determine optimal concentration

  • Washing stringency: Increase TBST (0.1% to 0.3% Tween-20) and wash duration

  • Pre-adsorption: Incubate antibody with extracts from knockout plants to remove cross-reactive antibodies

• Validation controls:

  • Include protein extracts from knockout plants as negative controls

  • Use recombinant AT2G27360 as positive control

  • Test antibody specificity in tissues with known differential expression
    This systematic approach helps distinguish true AT2G27360 signal from artifacts, particularly important when studying closely related GDSL lipase family members .

How can researchers overcome challenges in detecting low-abundance AT2G27360 protein in specific tissues or conditions?

For enhanced detection of low-abundance AT2G27360 protein, implement this methodological approach:

• Sample enrichment techniques:

  • Subcellular fractionation focusing on endomembrane components

  • Immunoprecipitation-based enrichment prior to analysis

  • Protein concentration methods (TCA precipitation, acetone precipitation)

• Detection sensitivity enhancement:

  • Signal amplification using tyramide signal amplification (TSA) for immunohistochemistry

  • Enhanced chemiluminescence (ECL) with extended exposure times for Western blots

  • Fluorescent secondary antibodies with appropriate filter sets for improved signal-to-noise ratio

• Protocol modifications:

  • Extended primary antibody incubation (48-72 hours at 4°C)

  • Increased protein loading (50-100 μg total protein)

  • Gradient gels for improved separation and concentration of target protein bands

  • Film exposure using a series of times from 30 seconds to overnight

• Alternative detection methods:

  • Targeted mass spectrometry using selected reaction monitoring (SRM)

  • Proximity ligation assay (PLA) for in situ detection

  • Protein array techniques for multiplexed analysis
    These approaches significantly improve detection sensitivity while maintaining specificity, enabling investigation of AT2G27360 expression in conditions where the protein is minimally expressed .