At1g25240 Antibody

What is the At1g25240 protein and why is it significant in research?

At1g25240 is a gene that encodes a putative clathrin assembly protein in Arabidopsis thaliana. The protein consists of 376 amino acids and is believed to play a crucial role in clathrin-mediated endocytosis and cellular trafficking pathways. Its significance in research stems from its involvement in fundamental cellular processes that regulate membrane dynamics and protein transport. Understanding this protein helps elucidate mechanisms of vesicular trafficking in plant cells, which impacts numerous physiological processes including hormone signaling, pathogen response, and nutrient uptake. The protein has the UniProt accession Q9FRH3 and goes by the full name "Putative clathrin assembly protein At1g25240."

What types of At1g25240 antibodies are currently available for research?

Based on available research tools, there are several types of antibodies targeting the At1g25240 protein. These include:

• N-terminal targeting antibodies (X-Q9FRH3-N): These are combinations of mouse monoclonal antibodies that recognize epitopes at the N-terminus of the protein.

• C-terminal targeting antibodies (X-Q9FRH3-C): These antibody combinations target the C-terminal region of the protein.

• Middle region targeting antibodies (X-Q9FRH3-M): These target non-terminal sequences within the protein.
  Each antibody type is available as a combination of individual monoclonal antibodies raised against synthetic peptide antigens from the corresponding regions of the target protein. These combinations can be used directly or deconvoluted into individual monoclonal antibodies after epitope determination if needed for specific applications.

What is the recommended methodology for validating a new At1g25240 antibody?

Validating a new At1g25240 antibody requires a systematic approach to ensure specificity and sensitivity. The methodology should include:

• Initial ELISA screening: Confirm antibody-antigen interaction with a titer of approximately 10,000, which corresponds to detection capability of around 1 ng of target protein on Western blots.

• Western blot validation: Test the antibody on plant tissue samples known to express At1g25240, alongside negative controls such as knockout mutants if available. Look for a single band at the expected molecular weight (approximately 42 kDa for the native protein).

• Immunoprecipitation: Confirm the antibody can pull down the native protein from plant cell lysates.

• Immunolocalization: Perform immunofluorescence microscopy to verify that the antibody localizes to expected cellular compartments, particularly to clathrin-coated vesicles and the plasma membrane.

• Cross-reactivity testing: Examine potential cross-reactivity with related proteins, especially other clathrin assembly proteins.
  The validation should include both wild-type plants and, if available, plants with altered expression of At1g25240 to confirm specificity of the antibody response.

How can At1g25240 antibodies be utilized in studying plant stress responses?

At1g25240 antibodies can be instrumental in investigating how clathrin-mediated endocytosis (CME) responds to various stress conditions in plants. The methodological approach involves:

• Stress treatment protocols: Subject plants to different stressors (drought, salt, pathogen exposure) using standardized protocols.

• Temporal analysis: Collect tissue samples at defined time points (0, 1, 3, 6, 12, 24 hours) after stress induction.

• Protein expression profiling: Use At1g25240 antibodies in Western blot analysis to quantify changes in protein abundance relative to control proteins (e.g., actin, tubulin).

• Subcellular localization changes: Employ immunofluorescence microscopy with the antibodies to track potential relocalization of the clathrin assembly protein during stress response.

• Co-immunoprecipitation: Use At1g25240 antibodies to pull down protein complexes under stress conditions, followed by mass spectrometry to identify stress-specific interaction partners.

• Phosphorylation state analysis: Combine At1g25240 antibodies with phospho-specific detection methods to determine if stress alters the post-translational modification status of the protein.
  This comprehensive approach allows researchers to understand how membrane trafficking adapts during stress responses, potentially revealing mechanisms for improving plant resilience.

What are the considerations for employing At1g25240 antibodies in comparative studies across plant species?

When using At1g25240 antibodies for cross-species studies, researchers should consider several methodological aspects:

• Sequence homology analysis: First, conduct bioinformatic analysis of protein sequence conservation across target species. For example, Momordica charantia (bitter melon) expresses a homologous putative clathrin assembly protein (LOC111023025) that may cross-react with Arabidopsis-derived antibodies.

• Epitope conservation assessment: Analyze specific epitope regions recognized by the antibodies to determine conservation across species. The sequence MKLWKRASGALKDRKTLFTIGFSRKT... should be compared with potential homologs in target species.

• Validation in each species: Perform Western blot analysis with positive and negative controls for each new species before proceeding with experimental applications.

• Titration optimization: Determine optimal antibody concentrations for each species, as binding affinity may vary due to sequence differences.

• Alternative detection strategies: Consider using multiple antibodies targeting different regions (N-terminal, C-terminal, and middle region) to increase detection reliability across species.

• Control experiments: Include recombinant protein controls from each species to calibrate detection sensitivity.
  This systematic approach ensures reliable comparative analysis of clathrin assembly proteins across plant lineages, potentially revealing evolutionary conservation or divergence of trafficking mechanisms.

How can At1g25240 antibodies be used in conjunction with advanced imaging techniques?

Integrating At1g25240 antibodies with cutting-edge imaging technologies enables sophisticated analysis of clathrin assembly dynamics in plant cells. The methodological framework includes:

• Super-resolution microscopy preparation:

  • Sample fixation: Optimize fixation protocols (4% paraformaldehyde, 20 minutes) to preserve native protein distribution

  • Permeabilization: Fine-tune cell wall digestion and membrane permeabilization to maximize antibody penetration while maintaining structural integrity

  • Blocking: Use specific blocking agents (3% BSA, 0.1% Triton X-100) to minimize background signal

• Structured illumination microscopy (SIM) protocol:

  • Primary antibody incubation: Apply At1g25240 antibodies at 1:500 dilution overnight at 4°C

  • Secondary antibody selection: Use fluorophore-conjugated antibodies optimized for SIM imaging

  • Multi-channel imaging: Include markers for plasma membrane and endosomal compartments

• Live-cell imaging approaches:

  • Antibody fragment generation: Produce Fab fragments from At1g25240 antibodies for live-cell applications

  • Conjugation strategies: Direct labeling with small fluorophores (Alexa Fluor 647) for minimal interference

  • Microinjection techniques: Deliver labeled antibody fragments into living plant cells

• Correlative light and electron microscopy (CLEM):

  • Immunogold labeling: Use At1g25240 antibodies with gold nanoparticle-conjugated secondary antibodies

  • Serial section preparation: Generate ultrathin sections (70-100 nm) of embedded plant tissue

  • Registration procedures: Align fluorescence and electron microscopy data for precise correlation
    This integrative approach allows visualization of clathrin assembly protein dynamics at unprecedented resolution, revealing the spatial organization and temporal dynamics of endocytic machinery in plant cells.

What are the optimal conditions for using At1g25240 antibodies in immunoprecipitation experiments?

For successful immunoprecipitation (IP) of At1g25240 protein complexes, researchers should follow these methodological guidelines:

• Tissue extraction buffer optimization:

  Component	Concentration	Purpose
  Tris-HCl pH 7.5	50 mM	Buffering
  NaCl	150 mM	Ionic strength
  EDTA	1 mM	Chelating agent
  EGTA	1 mM	Calcium chelator
  NP-40 or Triton X-100	0.5-1%	Membrane solubilization
  Protease inhibitors	1×	Prevent degradation
  Phosphatase inhibitors	1×	Preserve phosphorylation
  DTT	1 mM	Reducing agent

• Antibody coupling strategies:

  • Direct coupling: Conjugate At1g25240 antibodies to activated agarose or magnetic beads (5 μg antibody per 25 μl bead volume)

  • Indirect coupling: Use Protein A/G beads to capture antibody-antigen complexes

  • Crosslinking: Apply DSS or BS3 crosslinkers to prevent antibody leaching during elution

• Incubation parameters:

  • Pre-clearing: Incubate lysate with beads alone for 1 hour at 4°C to reduce non-specific binding

  • Immunocapture: Mix lysate with antibody-coupled beads for 4 hours or overnight at 4°C with gentle rotation

  • Washing stringency: Perform 4-5 washes with decreasing detergent concentrations (0.5% to 0.1%)

• Elution methods:

  • Denaturing: Use SDS sample buffer at 95°C for 5 minutes

  • Native: Apply excess antigen peptide (0.1-0.5 mg/ml) or low pH glycine buffer (pH 2.5)

• Troubleshooting strategies:

  • Low yield: Increase starting material or extend incubation time

  • High background: Add additional pre-clearing steps or increase wash stringency

  • Protein degradation: Supplement with additional protease inhibitors and work at 4°C throughout
    This detailed protocol establishes conditions that preserve native protein interactions while minimizing non-specific binding, enabling reliable analysis of At1g25240-associated protein complexes.

How can researchers address antibody cross-reactivity issues when working with At1g25240 antibodies?

Cross-reactivity is a common challenge when working with antibodies against plant proteins. For At1g25240 antibodies, researchers can employ these methodological approaches to address specificity concerns:

• Absorption controls:

  • Pre-absorb antibodies with the immunizing peptide (10-50 μg/ml) to confirm signal specificity

  • Perform parallel experiments with absorbed and non-absorbed antibody preparations

  • Compare signal reduction patterns to identify potential cross-reactive epitopes

• Genetic validation strategies:

  • Test antibodies on At1g25240 knockout/knockdown lines as negative controls

  • Use overexpression lines as positive controls with enhanced signal

  • Employ CRISPR-edited plant lines with epitope modifications to confirm binding sites

• Cross-species testing protocol:

  • Analyze antibody performance in species with known sequence differences in the target region

  • Create a cross-reactivity profile based on sequence homology and experimental validation

  • Use this data to predict potential false positives in experimental systems

• Epitope mapping approach:

  • Generate a peptide array covering the full At1g25240 sequence

  • Test antibody binding to each peptide to identify specific recognition motifs

  • Compare these motifs against the plant proteome to identify potential cross-reactive proteins

• Signal validation matrix:

  Validation Method	Expected Result for Specific Signal	Potential Cross-Reactivity Indicator
  Western blot size	Single band at 42 kDa	Additional bands at unexpected sizes
  Peptide competition	Complete signal abolishment	Partial signal reduction
  Knockout control	No signal	Persistent signal
  Orthogonal antibodies	Identical localization pattern	Divergent localization patterns
  Mass spectrometry	Confirmation of target identity	Identification of unexpected proteins

By implementing this comprehensive cross-reactivity assessment framework, researchers can establish confidence in the specificity of their At1g25240 antibody signals and develop strategies to mitigate any identified issues.

What are the recommended protocols for quantifying At1g25240 protein levels using antibody-based methods?

Accurate quantification of At1g25240 protein levels requires rigorous methodological approaches. Researchers should implement the following protocols:

• Western blot quantification:

  • Sample preparation: Standardize tissue collection, extraction buffer composition, and protein determination methods

  • Loading controls: Include both housekeeping proteins (actin, tubulin) and total protein stains (Ponceau S, SYPRO Ruby)

  • Technical replication: Run 3-4 technical replicates per biological sample

  • Standard curve generation: Include a dilution series of recombinant At1g25240 protein (0.1-10 ng)

  • Imaging parameters: Use a linear dynamic range detection system and avoid signal saturation

  • Analysis software: Apply ImageJ or similar tools with background subtraction and normalization

• Enzyme-linked immunosorbent assay (ELISA):

  • Format selection: Sandwich ELISA using capture antibodies targeting different epitopes than detection antibodies

  • Standard curve preparation: Use purified recombinant At1g25240 protein (1-1000 ng/ml)

  • Sample dilution series: Test multiple dilutions to ensure readings fall within the linear range

  • Antibody optimization: Determine optimal concentrations through checkerboard titration

  • Data analysis: Apply four-parameter logistic regression for standard curve fitting

• Flow cytometry:

  • Cell preparation: Optimize protoplast isolation to maintain protein integrity

  • Fixation and permeabilization: Test multiple fixatives (paraformaldehyde, methanol) and permeabilization agents

  • Antibody labeling: Use directly conjugated antibodies or minimal fluorophore:antibody ratios

  • Controls: Include isotype controls and competition with immunizing peptide

  • Analysis parameters: Set consistent gating strategies across experiments

• Quantitative multiplexed immunofluorescence:

  • Target multiplexing: Combine At1g25240 antibody with markers for subcellular compartments

  • Signal calibration: Include calibration beads with known fluorophore numbers

  • Image acquisition: Standardize exposure settings, detector gain, and optical configurations

  • Quantification: Apply automated segmentation and intensity measurement algorithms
    This systematic approach to protein quantification enables reliable measurement of At1g25240 expression levels across experimental conditions, providing robust data for comparative analyses.

How should researchers interpret differences in At1g25240 antibody binding patterns across developmental stages?

When analyzing developmental changes in At1g25240 protein expression or localization, researchers should apply the following interpretive framework:

• Developmental expression profile analysis:

  • Establish a comprehensive timeline of At1g25240 expression across key developmental stages

  • Normalize protein levels to appropriate stage-specific reference proteins

  • Distinguish between changes in total protein abundance versus shifts in subcellular distribution

  • Consider tissue-specific expression patterns that may emerge during development

• Correlation with developmental markers:

  • Analyze co-expression patterns with known developmental regulators

  • Plot At1g25240 levels against established developmental milestones

  • Test for statistical significance of expression changes at developmental transitions

• Functional interpretation strategies:

  • Connect expression patterns to corresponding developmental processes requiring vesicular trafficking

  • Evaluate whether changes represent constitutive adaptation or responsive regulation

  • Consider post-translational modifications that may affect antibody recognition during development

• Common interpretation pitfalls:

  • Avoid attributing all binding pattern changes to altered expression (consider epitope masking)

  • Account for tissue composition changes that may affect apparent expression levels

  • Control for developmental changes in cell wall/membrane permeability affecting antibody accessibility

• Integrative data visualization:

  Developmental Stage	At1g25240 Level	Subcellular Localization	Associated Developmental Processes
  Seed germination	Moderate	Primarily plasma membrane	Cell elongation, vesicle trafficking
  Seedling growth	High	Plasma membrane and early endosomes	Auxin transport, nutrient uptake
  Vegetative growth	Moderate	Distributed in vesicular patterns	Maintenance of cell polarity
  Flowering transition	Variable	Tissue-dependent redistribution	Reproductive organ development
  Senescence	Decreasing	Predominantly vacuolar	Cellular remodeling, nutrient recycling

This comprehensive analytical approach helps distinguish between biologically meaningful changes in At1g25240 function during development and technical artifacts that may arise from tissue-specific factors affecting antibody performance.

How can researchers integrate At1g25240 antibody data with other -omics datasets for systems biology approaches?

Integrating antibody-based protein data with other -omics datasets enables comprehensive systems-level understanding of At1g25240 function. Researchers should implement the following integration methodology:

• Multi-omics data acquisition planning:

  • Coordinated sampling: Collect material for different -omics analyses from the same experimental units

  • Temporal alignment: Synchronize sampling timepoints across platforms

  • Metadata standardization: Maintain consistent experimental annotation across all datasets

• Data preprocessing harmonization:

  • Normalization strategies: Apply platform-appropriate normalization while preserving biological variation

  • Missing value handling: Implement consistent approaches across datasets (imputation or filtering)

  • Batch effect correction: Use ComBat, SVA, or related algorithms to minimize technical variation

• Cross-platform correlation analysis:

  • Pairwise correlation: Calculate Pearson/Spearman correlations between At1g25240 protein levels and transcript abundance

  • Time-lagged correlation: Apply dynamic time warping for temporal datasets

  • Multi-level correlation: Analyze relationships across protein, transcript, metabolite, and phenotype levels

• Network integration approaches:

  • Protein-centric networks: Build interaction networks with At1g25240 as a focal point

  • Co-expression modules: Identify modules showing coordinated regulation with At1g25240

  • Pathway enrichment: Map integrated data to known biological pathways and cellular processes

• Integrative visualization strategies:

  Integration Level	Recommended Visualization	Analytical Output
  Protein-transcript	Scatter plots with regression	Correlation metrics, discordance identification
  Temporal dynamics	Heatmaps with hierarchical clustering	Temporal response patterns, co-regulated genes
  Pathway mapping	Overlay diagrams on cellular pathways	Functional context, regulatory relationships
  Network visualization	Force-directed graphs	Protein-protein interactions, regulatory hubs
  Multi-omics integration	Circos plots or Sankey diagrams	System-wide relationships, information flow

• Causal inference methods:

  • Bayesian networks: Infer directional relationships between molecular components

  • Structural equation modeling: Test hypothesized causal structures

  • Granger causality: Assess temporal precedence in time-series data This integrated systems biology approach contextualizes At1g25240 antibody data within the broader cellular machinery, revealing functional relationships and regulatory mechanisms that would not be apparent from protein analysis alone.