ASHH2 Antibody

What is ASHH2 and why are antibodies against it important for epigenetic research?

ASHH2 (also called SDG8, EFS, and CCR1) is a histone lysine methyltransferase (HMTase) that plays a crucial role in defining transcriptionally permissive or repressive chromatin states . It is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase . Antibodies against ASHH2 are important for epigenetic research because they allow scientists to study histone modification patterns, particularly H3K36 trimethylation, which ASHH2 catalyzes.

In plant models like Arabidopsis, ASHH2 regulates numerous developmental processes including reproduction, with mutation of the gene resulting in pleiotropic effects . ChIP experiments using ASHH2 antibodies have demonstrated that genes down-regulated in ashh2 mutants show reduction specifically in H3K36 trimethylation but not in H3K4me3 or H3K36me2 . This indicates ASHH2's specific role in H3K36 trimethylation, making antibodies against this protein valuable tools for studying this particular epigenetic mark.

For successful research applications, it's important to distinguish ASHH2 from the similarly named but functionally distinct proteins ASH2 (a component of the SET1/ASH2 histone methyltransferase complex that methylates K4 of histone H3) and ASAH2 (an unrelated human protein) .

What are the primary applications of ASHH2 antibodies in research settings?

ASHH2 antibodies are versatile tools employed in several key research applications:

Chromatin Immunoprecipitation (ChIP): The predominant application of ASHH2 antibodies is in ChIP assays to identify genomic regions where ASHH2 binds and potentially modifies histones. From the provided search results, researchers have successfully used ChIP with H3K36me3 antibodies to demonstrate ASHH2's role in depositing this specific histone mark at genes like AP1, AtDMC1, and MYB99 .

Immunoblotting/Western Blotting: ASHH2 antibodies can be used to detect and quantify ASHH2 protein levels in different tissue samples or experimental conditions, with typical working dilutions ranging from 0.04-0.4 μg/mL (based on recommendations for similar antibodies) .

Immunofluorescence: For subcellular localization studies, ASHH2 antibodies enable visualization of the protein within cells, with recommended concentrations of 0.25-2 μg/mL .

Immunohistochemistry: ASHH2 antibodies can be used to examine protein expression in tissue sections, typically at dilutions of 1:1000-1:2500 .

The specific application dictates antibody selection, with considerations for formulation (e.g., buffered aqueous glycerol solution), species reactivity, and whether a primary or conjugated antibody is required .

How should researchers validate the specificity of ASHH2 antibodies?

Validating antibody specificity is critical for ensuring reliable research results. For ASHH2 antibodies, researchers should implement the following validation protocol:

Positive and Negative Controls: Include wild-type samples (positive control) alongside ashh2 mutant samples (negative control) when available . The dramatic reduction in signal in mutant samples provides strong evidence of antibody specificity.

Western Blot Analysis: Perform immunoblotting to confirm that the antibody detects a protein of the expected molecular weight for ASHH2. This should reveal a single predominant band at the predicted size, with minimal cross-reactivity .

Peptide Competition Assay: Pre-incubate the antibody with the immunogen peptide (such as recombinant ASHH2 protein or the epitope peptide used to generate the antibody) . If the antibody is specific, pre-incubation should abolish or significantly reduce signal in subsequent applications.

Cross-Reactivity Testing: Test the antibody against related proteins, particularly ASH2 and other SET-domain proteins, to ensure it doesn't cross-react with these similar proteins .

Multiple Antibody Verification: When possible, verify findings using multiple antibodies targeting different epitopes of ASHH2 to rule out epitope-specific artifacts.

Functional Validation: For ChIP applications specifically, validate that the antibody can detect changes in H3K36 trimethylation in genes known to be regulated by ASHH2, such as AP1, AtDMC1, and MYB99 .

What is the relationship between ASHH2 antibodies and histone modification detection?

While ASHH2 antibodies directly detect the ASHH2 protein itself, they are often used in conjunction with antibodies against specific histone modifications to establish the functional relationship between ASHH2 and its epigenetic effects:

Complementary Antibody Usage: Research protocols typically employ ASHH2 antibodies alongside antibodies against H3K36me3, H3K36me2, and H3K4me3 to provide a comprehensive picture of histone modification landscapes . This combinatorial approach allows researchers to correlate ASHH2 binding with specific histone marks.

Sequential ChIP (Re-ChIP): This technique involves performing successive immunoprecipitations with ASHH2 antibodies followed by histone modification antibodies (or vice versa). This determines whether ASHH2 and specific histone marks co-occur at the same genomic locations.

Causality Assessment: In experimental designs involving ashh2 mutants, researchers can use histone modification antibodies to demonstrate cause-effect relationships. For instance, studies have shown that H3K36me3 (but not H3K4me3 or H3K36me2) is specifically reduced at certain gene loci in ashh2 mutants, establishing ASHH2 as an H3K36 trimethyltransferase .

Temporal Studies: By combining ASHH2 antibodies with histone modification antibodies in time-course experiments, researchers can determine the sequence of events in epigenetic regulation—whether ASHH2 binding precedes or follows certain histone modifications.

This multi-antibody approach has revealed that ASHH2 specifically affects H3K36 trimethylation at target genes involved in floral organ identity, embryo sac development, and pollen development, demonstrating its specialized role in reproductive development .

How can ASHH2 antibodies be optimized for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ASHH2 antibodies for ChIP requires careful attention to several experimental parameters:

Fixation Conditions: For plant tissues containing ASHH2, optimal crosslinking is typically achieved with 1% formaldehyde for 10-15 minutes at room temperature. Over-fixation can mask epitopes and reduce antibody accessibility, while under-fixation may fail to preserve protein-DNA interactions.

Chromatin Fragmentation: For ASHH2 ChIP, aim for chromatin fragments between 200-500bp. Sonication parameters should be optimized for each tissue type, with careful monitoring via gel electrophoresis to ensure appropriate fragmentation.

Antibody Concentration: Titrate ASHH2 antibodies to determine optimal concentrations. Based on the data from similar antibodies, a starting range of 2-5 μg per ChIP reaction is recommended , but this should be empirically determined for each experimental system.

Incubation Parameters: Extend antibody-chromatin incubation to overnight at 4°C with gentle rotation to maximize binding, especially when studying low-abundance targets like ASHH2 in specific tissues.

Washing Stringency: Optimize wash buffers to minimize background while maintaining specific signal. For ASHH2 ChIP, a graduated washing strategy is recommended, starting with low-stringency buffers and progressing to higher stringency.

Controls: Include both technical controls (IgG negative control, input sample) and biological controls (known ASHH2 target genes such as AP1, AtDMC1, and MYB99 as positive controls) . Analysis of H3K36me3 levels at these loci can serve as functional validation.

Sonication-Resistant Regions: Be aware that heterochromatic regions may be resistant to sonication and require additional optimization strategies, such as increased sonication cycles or alternative chromatin preparation methods.

For specialized applications like sequential ChIP to analyze ASHH2 and H3K36me3 co-occurrence, additional protocol modifications including gentler elution conditions between immunoprecipitations are necessary.

What are the key considerations for using ASHH2 antibodies in different model organisms?

Using ASHH2 antibodies across different model organisms requires careful attention to epitope conservation, species reactivity, and model-specific optimization:

Epitope Conservation Analysis:
Prior to selecting an ASHH2 antibody for cross-species applications, researchers should perform sequence alignment of the immunogen sequence against the target organism's ASHH2 homolog. The immunogen sequence "RTFGDVLQPAKPEYRVGEVAEVIFVGANPKNSVQNQNHQT" (or similar sequences used for other antibodies) should be checked for conservation in the target species.

Species-Specific Validation:
Each model organism requires independent validation of antibody specificity:

• Arabidopsis: The model system where ASHH2 function is best characterized . Validation can include testing in ashh2 mutant lines versus wild-type.

• Mammals: Human ASH2 differs substantially from plant ASHH2, so antibodies raised against human ASH2 (like the one described in result ) won't recognize plant ASHH2.

• Other plant species: When extending to crop plants or other model plant systems, pilot experiments with western blots are essential before proceeding to more complex applications.

Tissue-Specific Considerations:
ASHH2 expression varies across tissues. In Arabidopsis, ASHH2 promoter-reporter gene expression was observed at the site of megaspore mother cell formation, tapetum layers, and pollen . Antibody applications should account for these expression patterns when sampling tissues or interpreting results.

Protocol Adaptations:
Model-specific modifications may include:

• Protein extraction buffers optimized for plant tissues with high polyphenol content

• Altered fixation protocols for tissues with different permeability characteristics

• Adjusted antibody concentrations based on target abundance in different species

Expected Phenotypic Correlations:
When validating ASHH2 antibodies in new model systems, researchers should correlate antibody signals with expected phenotypes. For instance, in Arabidopsis, ashh2 mutations lead to reproductive defects including reduced pollen production and embryo sac development , which should correlate with ASHH2 protein localization patterns.

How can researchers troubleshoot non-specific binding and background issues with ASHH2 antibodies?

Non-specific binding and background issues can significantly impact experimental outcomes when working with ASHH2 antibodies. Here's a systematic troubleshooting approach:

Sources of Background in Immunoblotting:

• Primary Antibody Concentration: Titrate down from manufacturer recommendations (starting at 0.04-0.4 μg/mL) if background is excessive.

• Blocking Optimization: Test alternative blocking agents (BSA, non-fat milk, commercial blockers) at varying concentrations (3-5%).

• Washing Stringency: Increase number and duration of washes, potentially adding 0.1-0.3% SDS to TBST for stubborn background.

• Sample Preparation: Ensure complete protein denaturation and consider pre-clearing lysates with Protein A/G beads.

Immunofluorescence/Immunohistochemistry Background Reduction:

• Autofluorescence Quenching: For plant tissues, which often exhibit high autofluorescence, pre-treat sections with sodium borohydride or employ spectral unmixing during imaging.

• Antibody Dilution Series: Test a wider range around the recommended 0.25-2 μg/mL for immunofluorescence.

• Secondary Antibody Controls: Always include secondary-only controls to identify non-specific binding of the detection system.

• Permeabilization Optimization: Adjust detergent concentration and incubation times for optimal antibody access with minimal structural disruption.

ChIP-Specific Background Reduction:

• Pre-blocking Antibodies: Incubate ASHH2 antibodies with non-specific DNA (e.g., sheared salmon sperm DNA) before adding to chromatin.

• Pre-clearing Chromatin: Pre-clear chromatin with Protein A/G beads to remove components that bind non-specifically.

• Sequential Washes: Implement graduated stringency washing protocols, increasing salt concentration with each wash.

• Comparison to IgG Control: Normalize ChIP data to IgG control from the same species as the primary antibody.

Experimental Design to Account for Background:

• Knockout/Knockdown Controls: Include ashh2 mutant samples as negative controls.

• Competition Assays: Pre-incubate antibody with immunizing peptide to confirm signal specificity.

• Multiple Antibody Validation: Confirm findings with a second antibody targeting a different ASHH2 epitope.

• Dual-Labeling Approaches: Co-localize ASHH2 with known interacting partners or histone marks like H3K36me3 .

A systematic approach to antibody dilution optimization and appropriate controls significantly enhances experimental reliability when working with ASHH2 antibodies.

What experimental designs can effectively study the relationship between ASHH2 and its histone methylation targets?

Advanced studies of ASHH2's relationship with histone methylation targets require sophisticated experimental designs:

ChIP-seq Comparative Analysis:
Design parallel ChIP-seq experiments using:

• ASHH2 antibodies to identify genomic binding sites

• H3K36me3 antibodies to map trimethylation patterns

• H3K36me2 antibodies as a control mark (not affected by ASHH2)

• H3K4me3 antibodies as another control mark (not affected by ASHH2)

This comprehensive approach allows for genome-wide correlation analysis between ASHH2 binding and specific methylation patterns. The search results indicate that genes down-regulated in ashh2 mutants show specific reduction in H3K36me3 but not in H3K4me3 or H3K36me2 .

Genetic Manipulation Coupled with ChIP:

This experimental matrix allows researchers to distinguish between ASHH2 binding and its enzymatic activity, providing insights into its mechanism of action.

Sequential ChIP (Re-ChIP):
This technique involves consecutive immunoprecipitations:

• First IP with ASHH2 antibody

• Second IP with H3K36me3 antibody (or vice versa)

This directly demonstrates co-occurrence of ASHH2 and H3K36me3 at specific genomic loci. The technique can be adapted for genome-wide analysis (Re-ChIP-seq).

Time-Course Experiments:
Using inducible ASHH2 expression systems:

• Induce ASHH2 expression

• Collect samples at defined time points (e.g., 0, 2, 4, 8, 24 hours)

• Perform ChIP for both ASHH2 and H3K36me3 at each time point

• Analyze temporal relationship between ASHH2 binding and H3K36me3 appearance

This reveals the kinetic relationship between ASHH2 recruitment and histone modification.

Target Gene Expression Analysis:
Correlate ChIP data with gene expression analysis:

• ChIP-seq to identify ASHH2 and H3K36me3 co-occupied genes

• RNA-seq or qRT-PCR of identified targets in wild-type vs. ashh2 mutants

This establishes the functional consequences of ASHH2-mediated histone methylation. Previous studies identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, including genes involved in floral organ identity, embryo sac, and anther/pollen development .

How do different ASHH2 antibody formats (monoclonal vs. polyclonal) compare in research applications?

The choice between monoclonal and polyclonal ASHH2 antibodies significantly impacts experimental outcomes and should be guided by the specific research application:

Polyclonal ASHH2 Antibodies:
Polyclonal antibodies, like those described in the search results , recognize multiple epitopes on the ASHH2 protein.

Advantages:

• Higher sensitivity due to recognition of multiple epitopes

• More robust to minor protein denaturation or modifications

• Better signal amplification in applications like immunohistochemistry

• Generally more effective for immunoprecipitation applications, including ChIP

Limitations:

• Batch-to-batch variability requires validation of each lot

• May exhibit higher background due to recognition of related epitopes

• Limited renewable supply from a single immunization

Best Applications:

• ChIP experiments where sensitivity is paramount

• Initial exploratory studies of ASHH2 localization and function

• Detection of ASHH2 in fixed tissues where epitope accessibility may be limited

Monoclonal ASHH2 Antibodies:
While not specifically mentioned in the search results, monoclonal antibodies against ASHH2 would recognize a single epitope.

Advantages:

• Consistent reproducibility between experiments and batches

• Higher specificity for a particular epitope

• Reduced background in applications like Western blotting

• Renewable source for long-term experimental consistency

Limitations:

• Potentially lower sensitivity than polyclonals

• More susceptible to epitope masking during fixation

• May be ineffective if the single epitope is modified or mutated

Best Applications:

• Quantitative Western blotting requiring high reproducibility

• Experiments comparing ASHH2 levels across multiple conditions

• Studies requiring absolute consistency over extended periods

Comparative Performance Table:

For ASHH2 research specifically, polyclonal antibodies may be advantageous for ChIP applications investigating H3K36 trimethylation patterns , while monoclonals might be preferable for precise quantification of ASHH2 protein levels across experimental conditions.

What are the current limitations of ASHH2 antibodies and how might researchers address them?

Current ASHH2 antibodies face several limitations that researchers should acknowledge and address:

Cross-Reactivity Challenges:
The nomenclature confusion between ASHH2, ASH2, and ASAH2 presents significant cross-reactivity risks . These proteins share partial sequence homology but have distinct functions: ASHH2 is involved in H3K36 trimethylation , while ASH2 is part of complexes that methylate H3K4 .

Solutions:

• Perform comprehensive validation using knockout/knockdown controls

• Conduct cross-reactivity testing against recombinant ASH2 and ASAH2 proteins

• Design experiments with orthogonal methods to confirm antibody specificity

• Consider custom antibody development against unique ASHH2 epitopes

Epitope Masking During Crosslinking:
For ChIP applications, formaldehyde crosslinking can mask ASHH2 epitopes, particularly in regions involved in protein-DNA or protein-protein interactions.

Solutions:

• Test native ChIP protocols that avoid formaldehyde fixation

• Optimize crosslinking conditions (reduced time or concentration)

• Evaluate alternative crosslinkers like DSG (disuccinimidyl glutarate)

• Use antibodies targeting different ASHH2 epitopes in parallel experiments

Post-Translational Modification Interference:
ASHH2 may undergo various post-translational modifications that could interfere with antibody recognition.

Solutions:

• Generate modification-specific antibodies when particular PTMs are of interest

• Verify antibody performance under conditions that alter PTM status

• Use phosphatase or deubiquitinase treatment controls when appropriate

• Combine immunoprecipitation with mass spectrometry to identify modifications

Model Organism Limitations:
Most commercially available ASHH2 antibodies are developed against human or Arabidopsis proteins, limiting cross-species applications.

Solutions:

• Perform sequence alignment to assess epitope conservation before application

• Validate antibodies specifically in each model organism

• Consider developing species-specific antibodies for novel model systems

• Use tagged ASHH2 constructs with well-characterized tag antibodies as alternatives

Technological Limitations Table:

By acknowledging these limitations and implementing appropriate technical strategies, researchers can enhance the reliability of their ASHH2 antibody-based experiments and advance our understanding of H3K36 trimethylation in gene regulation .