ASHR3 Antibody

What is ASHR3 and why is it important in plant research?

ASHR3 is a SET-domain protein that functions as a histone lysine methyltransferase, responsible for mono-, di-, or trimethylation of various lysine residues on N-terminal histone tails. It is particularly expressed in the root stem cell niche and contributes to coordinated divisions of daughter and grand-daughter cells . The importance of ASHR3 in plant research stems from its critical role in the maintenance of meristematic cell divisions and its involvement in epigenetic regulation through histone modification. Studies with the ashr3-1 mutant have revealed distorted patterns of replication and cell division, highlighting ASHR3's significance in proper root development . Current evidence indicates that ASHR3 is the first SET-domain protein identified with histone H3K36 mono-methyltransferase activity, making it a crucial research target for understanding epigenetic regulation in plants .

What validation methods should be used to confirm ASHR3 antibody specificity?

When validating ASHR3 antibodies, researchers should employ multiple complementary techniques:

• Western Blotting: Compare protein expression patterns between wild-type and ashr3 mutant plants. A specific antibody should show reduced or absent signal in the mutant.

• Immunohistochemistry (IHC): Perform parallel staining of wild-type and mutant tissues. Examine signal localization in the root stem cell niche where ASHR3 is known to be expressed .

• Chromatin Immunoprecipitation (ChIP): Validate antibody specificity by conducting ChIP on known ASHR3 targets followed by qPCR. The E2F binding site regions in the ASHR3 promoter can serve as positive controls .

• Immunofluorescence (IF): Compare cellular localization patterns between control and experimental samples, ensuring they match the expected nuclear localization of a histone methyltransferase.

• Peptide Competition Assay: Pre-incubate the antibody with excess ASHR3 peptide antigen before application. A specific antibody will show diminished signal.

For polyclonal antibodies, similar validation approaches to those used for other research antibodies can be applied, ensuring the most rigorous levels of quality and reproducibility .

What are the optimal sample preparation protocols for ASHR3 antibody applications?

For optimal results with ASHR3 antibodies in plant tissue, follow these sample preparation guidelines:

For Western Blotting:

• Harvest fresh root tissue and immediately flash-freeze in liquid nitrogen

• Grind tissue in extraction buffer containing protease inhibitors, phosphatase inhibitors, and deacetylase inhibitors

• Include 20mM N-ethylmaleimide to preserve protein methylation status

• Use a moderate detergent concentration (0.1-0.5% NP-40 or Triton X-100) to extract nuclear proteins

• Carefully control protein loading (20-40 μg total protein) for consistent results

For Immunohistochemistry/Immunofluorescence:

• Fix tissues in 4% paraformaldehyde for 1-2 hours at room temperature

• Perform antigen retrieval (10mM sodium citrate buffer, pH 6.0) to expose histone epitopes

• Use extended blocking (2-3 hours) with 3-5% BSA to reduce background

• Incubate with primary antibody at 4°C overnight

• Include appropriate negative controls (secondary antibody only, pre-immune serum)

For ChIP applications:

• Cross-link fresh tissue with 1% formaldehyde for 10 minutes

• Quench with 0.125M glycine

• Isolate and sonicate chromatin to 200-500bp fragments

• Pre-clear chromatin with protein A/G beads before antibody incubation

• Use 3-5 μg antibody per immunoprecipitation reaction

These protocols should be optimized based on specific plant tissues and experimental conditions.

What experimental controls are essential when using ASHR3 antibodies?

When conducting experiments with ASHR3 antibodies, the following controls are essential:

Positive Controls:

• Wild-type plant tissue known to express ASHR3, particularly from root meristematic zones

• Recombinant ASHR3 protein (if available)

• Tissues with E2Fa overexpression, as E2Fa regulates ASHR3 expression

Negative Controls:

• ashr3-1 mutant tissue samples (ideally the complete knockout)

• Secondary antibody-only control

• Isotype control antibody (same species and isotype as the ASHR3 antibody)

• Pre-immune serum control

• e2fa-2 e2fb-1 double mutant tissue (showing reduced ASHR3 expression)

Internal Controls:

• Antibody against housekeeping proteins (e.g., actin, tubulin) for loading controls in Western blots

• DAPI staining for nuclear localization in immunofluorescence

• Antibodies against other histone marks with known patterns for comparative ChIP experiments

For measuring ASHR3 enzymatic activity, parallel H3K36me1 and H3K36me2 assessments should be conducted, as ASHR3 has been associated with both mono-methylation and potential di-methylation activities .

How can ASHR3 antibodies be used to investigate cell cycle-dependent histone modifications?

ASHR3 antibodies can be powerful tools for studying cell cycle-dependent histone modifications, particularly given ASHR3's role as a direct target of E2F transcription factors that control S-phase dependent gene expression . Consider the following methodological approach:

Experimental Design:

• Cell Synchronization: Synchronize plant cells using hydroxyurea or aphidicolin treatment to arrest cells at specific cell cycle phases

• Time-course Analysis: Release cells from synchronization and collect samples at defined intervals

• Dual Immunostaining: Combine ASHR3 antibodies with cell cycle markers (e.g., CYCB1;1-GUS for G2/M phase)

• ChIP-seq Analysis: Perform ChIP-seq using both ASHR3 antibodies and H3K36me1/me2-specific antibodies across cell cycle stages

Data Collection and Analysis:

• Quantify ASHR3 protein levels throughout cell cycle progression

• Map genome-wide H3K36me1 and H3K36me2 distribution changes during cell cycle

• Correlate ASHR3 localization with replication timing domains

• Compare results between wild-type and ashr3-1 mutant lines

This approach can reveal how ASHR3-mediated histone methylation changes during the cell cycle and how it correlates with the coordinated cell division patterns observed in the root meristematic zone . The data may be presented as follows:

What approaches can resolve contradictory results when studying ASHR3's impact on histone methylation patterns?

When faced with contradictory results regarding ASHR3's impact on histone methylation, a systematic troubleshooting approach is essential:

Source Verification:

• Verify antibody specificity using multiple validation methods (Western blot, ChIP-qPCR)

• Confirm genetic identity of plant lines using genotyping

• Validate ashr3-1 mutant phenotype through established markers (e.g., root growth, EdU incorporation patterns)

Methodological Refinement:

• Cross-methodology Validation: Compare results using different techniques:

  • ChIP-seq vs. CUT&RUN for genome-wide methylation profiles

  • Mass spectrometry for direct measurement of histone modifications

  • Immunofluorescence for spatial distribution within tissues

• Genetic Complementation: Reintroduce wild-type ASHR3 into ashr3-1 mutants to confirm phenotype rescue

• Enzyme Activity Assay: Develop in vitro histone methyltransferase assays using:

  • Recombinant ASHR3 protein

  • Synthetic histone H3 peptides as substrates

  • Mass spectrometry to quantify methylation products

Data Reconciliation:

• Distinguish between direct and indirect effects through:

  • Inducible ASHR3 expression systems

  • Time-course studies following ASHR3 induction

  • Targeted mutations in the SET domain to separate enzymatic and structural roles

• Explore context-dependent activity by examining:

  • Different tissue types

  • Developmental stages

  • Environmental conditions

  • Cell cycle phases

The residual H3K36me1 present in ashr3-1 mutants suggests redundancy with other methyltransferases . Identify and characterize these enzymes (potentially other ASHH and ASHR proteins) to develop a comprehensive model of H3K36 methylation regulation.

How can advanced microscopy techniques enhance the study of ASHR3 function in root development?

Advanced microscopy techniques can significantly expand our understanding of ASHR3 function in root development, particularly given its role in coordinating cell divisions in the root meristematic zone . Consider these methodological approaches:

Live Cell Imaging:

• 4D Confocal Imaging: Generate ASHR3-fluorescent protein fusions and image living roots over time to track:

  • Protein localization during cell cycle

  • Dynamic association with chromatin

  • Co-localization with replication machinery

• FRAP (Fluorescence Recovery After Photobleaching): Assess ASHR3 mobility and chromatin binding dynamics in different cell types of the root

• Light-Sheet Microscopy: For whole-organ imaging with minimal phototoxicity, allowing extended time-lapse experiments of root development

Super-Resolution Microscopy:

• STED or STORM Microscopy: Achieve sub-diffraction resolution to visualize:

  • Precise nuclear distribution of ASHR3

  • Association with specific chromatin domains

  • Co-localization with H3K36me1/me2 marks

• Expansion Microscopy: Physically expand the specimen to improve resolution of dense chromatin structures

Correlative Approaches:

• CLEM (Correlative Light and Electron Microscopy): Combine fluorescence imaging of ASHR3 with ultrastructural information about chromatin organization

• Multi-modal Imaging: Integrate:

  • EdU incorporation for DNA replication (as described in the original research)

  • CYCB1;1-GUS staining for mitotic cells

  • KNOLLE immunolocalization for cytokinesis

  • ASHR3 antibody staining for protein localization

Quantitative Analysis:

• Develop computational pipelines to quantify:

  • Cell division patterns and synchrony

  • Nuclear ASHR3 distribution

  • Correlation between ASHR3 levels and cell cycle progression

  • 3D chromatin organization changes in ashr3-1 mutants

These advanced imaging approaches can provide spatiotemporal information about ASHR3 function that complements molecular and genetic analyses, offering insights into how this histone methyltransferase coordinates cell division patterns in root development.

What are the emerging computational approaches for designing antibodies against targets like ASHR3?

Recent advances in AI-based technologies offer promising computational approaches for designing antibodies against targets like ASHR3:

AI-Based Antibody Generation:

• Language Model Approaches: Large language models like IgLM can be applied to generate de novo antibody sequences, treating protein sequences as a language with patterns that can be learned and generated .

• Structure-Based Design: Computational tools can model the structure of both the target antigen (ASHR3) and potential antibody candidates, predicting their interaction:

  • Generate diverse complementarity determining region 3 (CDRH3) sequences

  • Model antibody heavy chain structures

  • Evaluate structural similarities to known effective antibodies

• Template-Based Generation: Using germline-based templates to generate antigen-specific antibody sequences:

  • Start with appropriate germline V-gene templates

  • Generate diverse CDRH3 sequences that maintain critical structural features

  • Down-select candidates based on predicted binding affinity

Validation and Selection Pipeline:

• Generate large pools (1000+) of candidate antibody sequences using AI tools

• Assess sequence diversity and structural properties computationally

• Filter candidates based on:

  • Predicted structural similarity to known antibody templates

  • Sequence uniqueness compared to existing antibodies

  • Predicted binding affinity and specificity

• Select a manageable subset (10-20) for experimental validation

The success rate of such computational approaches has shown promise, with studies reporting ~15% hit rates for antigen-specific antibodies . This represents a significant improvement over traditional discovery methods and could accelerate the development of specific antibodies against targets like ASHR3.

These computational approaches offer efficient alternatives to traditional antibody discovery methods, which typically require complex experimental protocols and access to source samples with previous exposure to the target .

What is the optimal protocol for using ASHR3 antibodies in ChIP experiments?

For optimal chromatin immunoprecipitation (ChIP) experiments using ASHR3 antibodies, follow this detailed protocol:

Sample Preparation:

• Harvest 1-2 grams of fresh root tissue from 6-7 day-old seedlings

• Cross-link proteins to DNA with 1% formaldehyde for 10 minutes under vacuum

• Quench with 0.125M glycine for 5 minutes

• Rinse thoroughly with cold PBS

• Flash-freeze in liquid nitrogen and store at -80°C until use

Chromatin Extraction:

• Grind tissue to fine powder in liquid nitrogen

• Resuspend in extraction buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS, 1mM PMSF, protease inhibitor cocktail)

• Filter through two layers of Miracloth

• Centrifuge at 3000g for 10 minutes at 4°C

• Wash nuclear pellet twice with extraction buffer

Chromatin Shearing:

• Resuspend nuclei in sonication buffer

• Sonicate to achieve DNA fragments of 200-500bp

• Centrifuge at 16,000g for 10 minutes at 4°C

• Save 10% of supernatant as input control

Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads for 1 hour at 4°C

• Add 3-5μg of ASHR3 antibody to pre-cleared chromatin

• Incubate overnight at 4°C with gentle rotation

• Add protein A/G beads and incubate for 3 hours at 4°C

• Wash beads sequentially with:

  • Low salt wash buffer (2x)

  • High salt wash buffer (2x)

  • LiCl wash buffer (2x)

  • TE buffer (2x)

DNA Recovery:

• Elute protein-DNA complexes with elution buffer (1% SDS, 0.1M NaHCO₃)

• Reverse cross-links (add NaCl to 0.2M, incubate at 65°C overnight)

• Treat with RNase A and Proteinase K

• Purify DNA using phenol-chloroform extraction or commercial kits

Analysis:

• Perform qPCR targeting known ASHR3-associated regions

• Include E2F binding sites in the ASHR3 promoter as positive controls

• For genome-wide analysis, prepare libraries for ChIP-seq

The protocol should be tailored based on the specific ASHR3 antibody characteristics, with particular attention to optimization of antibody concentration and incubation conditions.

How can ASHR3 antibodies be used to study alterations in chromatin structure during plant stress responses?

ASHR3 antibodies can be instrumental in investigating chromatin remodeling during plant stress responses, given ASHR3's role in histone methylation. Here's a comprehensive methodological approach:

Experimental Design:

• Stress Treatment System:

  • Expose plants to defined stressors (drought, salt, heat, pathogen)

  • Include time-course sampling (0, 1, 3, 6, 12, 24 hours)

  • Maintain parallel control plants

• Multi-omics Integration:

  • ChIP-seq with ASHR3 antibodies

  • H3K36me1/me2 ChIP-seq

  • RNA-seq for transcriptome analysis

  • ATAC-seq for chromatin accessibility

• Cell-Type Specific Analysis:

  • Use FACS to isolate specific root cell populations

  • Analyze cell-type specific chromatin modifications

  • Compare meristematic vs. differentiated cells

Key Methodological Steps:

• Differential ChIP Analysis:

  • Compare ASHR3 binding profiles between stress and control conditions

  • Identify stress-responsive genomic regions with altered ASHR3 occupancy

  • Correlate with changes in H3K36me1/me2 distribution

• Combined IF-FISH:

  • Perform immunofluorescence with ASHR3 antibodies

  • Combine with fluorescent in situ hybridization for specific genomic loci

  • Visualize spatiotemporal changes in nuclear organization

• Genetic Interaction Analysis:

  • Compare wild-type vs. ashr3-1 stress responses

  • Examine other SET-domain mutants in parallel

  • Generate double mutants with stress-responsive transcription factors

Data Analysis Framework:

• Identify differentially methylated regions (DMRs) in response to stress

• Correlate DMRs with transcriptional changes

• Analyze gene ontology enrichment in affected regions

• Compare patterns across different stresses to identify common and unique responses

• Develop predictive models for ASHR3-dependent stress responses

This methodology would reveal how ASHR3-mediated H3K36 methylation contributes to chromatin reorganization during stress adaptation, potentially identifying novel stress response mechanisms operating at the epigenetic level.

What are the best strategies for multiplexed detection of ASHR3 and related histone modifications?

For comprehensive characterization of ASHR3 and associated histone modifications, multiplexed detection approaches offer significant advantages. Here are optimized strategies:

Multi-parameter Flow Cytometry:

• Sample Preparation:

  • Isolate nuclei from root tissues

  • Fix with 1% formaldehyde

  • Permeabilize with 0.1% Triton X-100

• Antibody Combinations:

  • ASHR3 antibody (conjugated to a distinct fluorophore)

  • Anti-H3K36me1 antibody (different fluorophore)

  • Anti-H3K36me2 antibody (different fluorophore)

  • Anti-H3 (total) as normalization control

  • DNA content stain (DAPI/PI) for cell cycle analysis

• Analysis Pipeline:

  • Gate on intact nuclei

  • Create biaxial plots of ASHR3 vs. each histone modification

  • Correlate with cell cycle phases

Multiplexed Immunofluorescence:

• Sequential Antibody Labeling:

  • Use tyramide signal amplification (TSA) for sequential detection

  • Apply ASHR3 antibody, followed by HRP-conjugated secondary

  • Develop with TSA-fluorophore 1

  • Quench HRP activity

  • Repeat with antibodies against H3K36me1, H3K36me2, etc.

• Spectral Imaging:

  • Capture full emission spectra at each pixel

  • Unmix overlapping fluorophore signals

  • Generate quantitative colocalization maps

Mass Cytometry (CyTOF):

• Metal-Conjugated Antibodies:

  • Label ASHR3 antibody with one metal isotope

  • Label modification-specific antibodies with distinct metals

  • Include markers for cell identity and cell cycle

• Single-Cell Analysis:

  • Measure dozens of parameters simultaneously

  • Create high-dimensional data visualization (tSNE/UMAP)

  • Identify cell subpopulations with distinct modification patterns

Sequential ChIP (Re-ChIP):

• Perform initial ChIP with ASHR3 antibody

• Elute under mild conditions

• Perform second ChIP on eluate using H3K36me1 antibody

• Analyze genomic regions bound by both ASHR3 and containing H3K36me1

These multiplexed approaches allow researchers to determine the spatiotemporal relationship between ASHR3 localization and its enzymatic products (H3K36me1/me2), providing insights into the dynamics of ASHR3 function in different cellular contexts.

How might single-cell technologies advance our understanding of ASHR3 function in plant development?

Single-cell technologies offer unprecedented opportunities to understand ASHR3 function at cellular resolution, particularly important given its role in coordinating cell divisions in root development . Here's how these approaches can be applied:

Single-Cell Epigenomics:

• Single-Cell ATAC-seq:

  • Isolate nuclei from root tissues

  • Generate cell-specific chromatin accessibility maps

  • Compare accessibility patterns between wild-type and ashr3-1 mutants

  • Identify cell type-specific regulatory elements affected by ASHR3

• Single-Cell CUT&Tag:

  • Use ASHR3 antibodies for direct profiling in single cells

  • Map H3K36me1/me2 distributions in individual cells

  • Correlate modifications with cell identity and cell cycle phase

• Single-Nucleus RNA-seq:

  • Profile transcriptomes of individual root cells

  • Identify gene expression changes in ashr3-1 mutant cells

  • Perform trajectory analysis to map developmental progressions

Integration and Analysis:

• Multi-modal Single-Cell Analysis:

  • Perform simultaneous protein (ASHR3) and RNA detection

  • Integrate with histone modification data

  • Create comprehensive cell atlases of root development

• Computational Trajectory Reconstruction:

  • Infer developmental trajectories from single-cell data

  • Map chromatin state changes along differentiation paths

  • Identify ASHR3-dependent decision points

• Spatial Single-Cell Mapping:

  • Combine single-cell data with spatial information

  • Create 3D maps of ASHR3 activity in the root

  • Correlate with patterns of coordinated cell divisions

The application of these technologies could reveal how ASHR3-mediated H3K36 methylation contributes to cell fate decisions, how it varies between cell types, and how it changes during development. This would significantly enhance our understanding of the molecular mechanisms underlying the coordinated cell division patterns observed in wild-type roots and their disruption in ashr3-1 mutants .

What are the considerations for developing new ASHR3 antibodies using AI-based design approaches?

Developing next-generation ASHR3 antibodies using AI-based design approaches requires careful consideration of several factors:

Target Selection and Antigen Design:

• Epitope Identification:

  • Analyze ASHR3 protein sequence for unique, accessible regions

  • Prioritize conserved functional domains (SET domain)

  • Use structural prediction to identify surface-exposed peptides

  • Consider post-translational modifications that may affect recognition

• Antigen Preparation:

  • Design recombinant proteins or synthetic peptides representing ASHR3

  • Express in bacterial/mammalian systems or synthesize chemically

  • Validate correct folding of recombinant proteins

AI-Based Antibody Generation:

• Model Selection:

  • Choose appropriate AI platforms (e.g., language models like IgLM)

  • Consider structure-based prediction tools

  • Evaluate models based on published validation criteria

• Sequence Generation Strategy:

  • Generate diverse complementarity determining region (CDR) sequences

  • Focus on CDRH3 for primary antigen recognition

  • Consider germline V-gene templates appropriate for the target

  • Generate 1,000+ candidate sequences for initial screening

• Computational Screening:

  • Model antibody structures to assess structural diversity

  • Calculate predicted binding affinities

  • Cluster candidates to ensure diversity

  • Select 10-20 top candidates for experimental validation

Experimental Validation Pipeline:

• Expression and Purification:

  • Express candidate antibodies in appropriate systems

  • Purify using standardized protocols

  • Verify structural integrity

• Binding Assays:

  • ELISA against recombinant ASHR3

  • Surface plasmon resonance for affinity measurement

  • Competitive binding assays

• Functional Validation:

  • Western blotting against plant extracts

  • Immunoprecipitation efficiency

  • ChIP performance

  • Compare against existing antibodies

The integration of AI-based design with rigorous experimental validation could yield antibodies with improved specificity, affinity, and application performance compared to traditionally generated antibodies . Expected success rates of ~15% for antigen-specific antibodies from computationally designed candidates are promising , suggesting this approach could efficiently produce valuable new tools for ASHR3 research.

How can researchers troubleshoot non-specific binding issues with ASHR3 antibodies?

When facing non-specific binding issues with ASHR3 antibodies, a systematic troubleshooting approach is essential:

Problem Identification:

• Characterize the Issue:

  • Multiple unexpected bands in Western blots

  • Non-nuclear staining in immunofluorescence

  • High background in immunohistochemistry

  • Enrichment of non-target regions in ChIP

• Control Analysis:

  • Compare signal in wild-type vs. ashr3-1 mutant tissues

  • Evaluate pre-immune serum results

  • Check secondary antibody-only controls

Optimization Strategies:

For Western Blotting:

• Buffer Optimization:

  • Increase blocking agent concentration (5% BSA or milk)

  • Add 0.1-0.5% Tween-20 to washing steps

  • Try different blocking agents (BSA, milk, commercial blockers)

• Antibody Conditions:

  • Titrate antibody concentration (try 1:500, 1:1000, 1:2000, 1:5000)

  • Reduce incubation time or temperature

  • Add competing proteins (1% BSA during antibody incubation)

• Sample Preparation:

  • Include additional protease inhibitors

  • Pre-clear lysates with Protein A/G beads

  • Use freshly prepared samples

For Immunohistochemistry/Immunofluorescence:

• Fixation Optimization:

  • Try different fixatives (paraformaldehyde, methanol, acetone)

  • Optimize fixation time

  • Test different antigen retrieval methods

• Blocking Enhancements:

  • Add normal serum from secondary antibody species

  • Include 0.1-0.3% Triton X-100 in blocking buffer

  • Extend blocking time to 2-3 hours

• Antibody Application:

  • Prepare antibody in fresh buffer immediately before use

  • Pre-absorb with plant tissue powder from ashr3-1 mutants

  • Reduce primary antibody concentration

For ChIP Applications:

• Chromatin Preparation:

  • Optimize sonication conditions

  • Pre-clear chromatin extensively

  • Use higher stringency wash buffers

• IP Conditions:

  • Reduce antibody amount

  • Shorten incubation time

  • Increase salt concentration in wash buffers

By systematically implementing these strategies and carefully documenting the results, researchers can identify optimal conditions for specific and reproducible detection of ASHR3 protein in their experimental systems.

What are the best approaches for quantitative analysis of ASHR3 expression and activity?

For rigorous quantitative analysis of ASHR3 expression and activity, researchers should implement these methodological approaches:

Quantitative Protein Analysis:

• Quantitative Western Blotting:

  • Use fluorescently-labeled secondary antibodies for wider linear range

  • Include recombinant ASHR3 standards at known concentrations

  • Normalize to multiple housekeeping proteins

  • Employ image analysis software with advanced quantification algorithms

• ELISA Development:

  • Design sandwich ELISA using purified ASHR3 antibodies

  • Generate standard curves with recombinant protein

  • Validate using wild-type and ashr3-1 mutant samples

  • Measure protein levels across tissues and developmental stages

• Mass Spectrometry:

  • Develop selected reaction monitoring (SRM) assays for ASHR3

  • Include isotopically labeled peptide standards

  • Measure absolute protein quantities

  • Simultaneously quantify H3K36me1/me2 levels

Activity Measurements:

• In Vitro Methyltransferase Assays:

  • Immunoprecipitate ASHR3 from plant tissues

  • Incubate with recombinant histone H3 and S-adenosyl methionine

  • Quantify methylation by:

    • Antibody detection of methylated products

    • Mass spectrometry of modified peptides

    • Incorporation of ³H-labeled methyl groups

• ChIP-qPCR for Target Occupancy:

  • Design primers for known ASHR3 binding regions

  • Calculate percent input or fold enrichment

  • Use spike-in controls for normalization

  • Compare enrichment patterns between conditions

• H3K36me1/me2 Global Levels:

  • Extract histones using acid extraction

  • Quantify modification levels by Western blotting

  • Normalize to total H3 levels

  • Compare wild-type and ashr3-1 mutants

Data Integration Framework:

• Correlate ASHR3 protein levels with H3K36me1/me2 abundance

• Develop mathematical models relating enzyme concentration to activity

• Integrate transcriptomic data to correlate histone modifications with gene expression

• Apply statistical methods to account for biological and technical variability

These approaches provide complementary data on ASHR3 expression and function, allowing researchers to build comprehensive quantitative models of ASHR3 activity in different biological contexts.