ASH2L Antibody

What is ASH2L and why is it significant in epigenetic research?

ASH2L is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. It is a core component of multimeric histone methyltransferase complexes involved in the maintenance of active transcription . ASH2L functions primarily by regulating histone H3 lysine 4 (H3K4) methylation, a critical epigenetic mark associated with active gene expression.

Research significance lies in ASH2L's crucial role in various biological processes:

• It stimulates the lysine methyltransferase (KMTase) activity of MLL1-4, SET1A, and SET1B when in complex with WDR5 and RbBP5

• It has been implicated in cancer development, particularly showing high expression in endometrial cancer with correlation to poor prognosis

• It demonstrates DNA-binding capability through its Forkhead-like helix-wing-helix (HWH) domain, contributing to gene-specific regulation

• It participates in transcriptional regulation through interactions with transcription factors like Ap2δ

What detection methods can be used with ASH2L antibodies in research?

ASH2L antibodies can be utilized across multiple experimental techniques to investigate its expression, localization, and function:

For optimal results, researchers should select antibodies validated for their specific application and include appropriate controls to distinguish specific from non-specific signals.

What are the critical epitopes to consider when selecting ASH2L antibodies?

When selecting ASH2L antibodies, researchers should consider targeting functionally relevant domains:

• The N-terminal region (Ash2L N) contains the DNA-binding HWH domain, critical for recruitment to specific genomic loci

• Key residues like Lys225 and Lys229 within the HWH domain are essential for DNA binding activity

• Regions mediating protein-protein interactions, such as those interacting with ERα-AF2 (282-595aa) domain

• The C-terminal region may be targeted for investigating protein complex formation

Antibodies recognizing these specific regions enable targeted investigation of different ASH2L functions. Researchers should verify whether the antibody recognizes the full-length protein or specific fragments, particularly when studying ASH2L mutants or truncated forms.

How should ASH2L antibodies be used in ChIP experiments?

Chromatin immunoprecipitation (ChIP) with ASH2L antibodies requires careful optimization:

Protocol overview:

• Cross-link protein-DNA complexes in cells with formaldehyde (typically 1%)

• Lyse cells and sonicate chromatin to fragments of approximately 200-500bp

• Immunoprecipitate with ASH2L antibody (either endogenous targeting or against epitope tags like Flag if using tagged constructs)

• Wash extensively to remove non-specific binding

• Reverse cross-links and purify DNA

• Analyze by qPCR, sequencing, or other methods

Research-based considerations:

• Flag-tagged ASH2L constructs have been successfully used for ChIP in erythroid cells to detect binding to the β-globin locus control region (LCR)

• The N-terminal region of ASH2L is sufficient for binding to specific genomic loci like the HS2 region

• Control experiments should include IgG controls and validation that ASH2L mutants (such as K225E and K229E) fail to bind target sites

• Consider dual ChIP to investigate co-occupancy with binding partners like transcription factors or other histone modifiers

What are the best practices for studying ASH2L's role in histone methyltransferase activity?

To investigate ASH2L's function in histone methyltransferase complexes:

• In vitro histone methyltransferase assays:

  • Immunoprecipitate ASH2L-containing complexes from nuclear extracts

  • Incubate with recombinant or native histone H3 and radiolabeled S-adenosyl methionine

  • Detect methylation by autoradiography or using methylation-specific antibodies

  • Include proper controls such as immunocomplexes with known HMT activity (e.g., anti-ALR, anti-Su(z)12)

  • Use H3 mutants (K4A, K9A, K27A) to determine specificity for H3K4 methylation

• Cellular studies:

  • Knockdown ASH2L using shRNA or CRISPR-Cas9

  • Rescue with wild-type or mutant ASH2L constructs

  • Examine H3K4 methylation levels by western blotting or ChIP

  • Quantify gene expression changes of known targets

  • Example: ASH2L knockdown in erythroid cells leads to decreased H3K4me3 and β-globin expression, which can be rescued by wild-type but not DNA-binding deficient ASH2L mutants

How can researchers investigate ASH2L's interaction with transcription factors?

ASH2L interactions with transcription factors can be studied through multiple approaches:

Co-immunoprecipitation:

• Express tagged versions of ASH2L and the transcription factor of interest

• Prepare nuclear extracts under non-denaturing conditions

• Immunoprecipitate using antibodies against either protein

• Detect interaction by western blotting

• Verify specificity by including unrelated nuclear proteins as negative controls

• Example: ASH2L specifically interacts with Ap2δ but not with Ap2α, -β, -γ, or -ε

Domain mapping:

• Generate truncated constructs of ASH2L and the transcription factor

• Perform GST pull-down experiments or co-immunoprecipitation

• Identify essential interaction domains

• Example: ASH2L mainly binds to ERα-AF2 (282-595aa) domain and not ERα-AF1

Functional validation:

• Establish reporter gene assays with promoters regulated by the transcription factor

• Co-express ASH2L and measure transcriptional activity

• Deplete endogenous ASH2L and assess impact on target gene expression

• Example: Ash2l increases transcriptional activity of Ap2δ in a dose-dependent manner

What are common issues with ASH2L antibodies in immunohistochemistry and how can they be resolved?

When using ASH2L antibodies for immunohistochemistry, researchers may encounter several challenges:

Challenge: High background staining

• Solution: Optimize antibody concentration through titration experiments

• Solution: Extend blocking steps using 3-5% BSA or serum from the species of secondary antibody

• Solution: Include additional washing steps with PBS containing 0.1-0.3% Tween-20

Challenge: Weak or absent signal

• Solution: Explore different antigen retrieval methods (heat-induced vs. enzymatic)

• Solution: Modify fixation protocol (duration and fixative concentration)

• Solution: Consider signal amplification systems

• Solution: Verify antigen preservation in samples through positive controls

Challenge: Inconsistent staining across samples

• Solution: Standardize tissue processing, fixation, and storage conditions

• Solution: Process all experimental samples in parallel

• Solution: Use automated staining systems when available

• Solution: Include internal reference tissues with known ASH2L expression levels

When evaluating ASH2L expression in endometrial tissues, researchers have successfully used immunohistochemistry with average optical density (AOD) measurements to quantify nuclear expression .

How can researchers optimize ASH2L antibody specificity in western blotting?

To improve specificity when using ASH2L antibodies in western blotting:

• Sample preparation optimization:

  • Use nuclear extraction protocols to enrich for ASH2L, which is predominantly nuclear

  • Include protease and phosphatase inhibitors to prevent degradation

  • Determine optimal protein loading amount (typically 20-50μg of nuclear extract)

• Antibody validation approaches:

  • Test antibody on samples with ASH2L knockdown or knockout

  • Compare results from multiple antibodies targeting different ASH2L epitopes

  • Include positive controls from tissues known to express ASH2L (e.g., endometrial cancer samples)

  • Verify expected molecular weight (~86 kDa for full-length ASH2L)

• Protocol refinements:

  • Optimize blocking conditions (5% non-fat milk vs. BSA)

  • Test different antibody dilutions and incubation conditions

  • Increase washing stringency to reduce non-specific binding

  • Consider using high-sensitivity, low-background detection systems

The integrity of ASH2L antibody has been demonstrated in studies comparing ASH2L expression between endometrial cancer tissues and benign endometrial tissues, showing significantly higher expression in cancer samples .

How should researchers analyze ASH2L ChIP-seq data to identify functional binding sites?

Analysis of ASH2L ChIP-seq data requires sophisticated computational approaches:

• Quality control and preprocessing:

  • Assess sequencing quality using FastQC

  • Filter low-quality reads and trim adapters

  • Align to reference genome using BWA or Bowtie2

  • Remove PCR duplicates to avoid bias

• Peak calling and annotation:

  • Use MACS2 or similar algorithms for peak identification

  • Compare to appropriate input control or IgG samples

  • Annotate peaks relative to genomic features (promoters, enhancers, etc.)

  • Focus on regions with strong ASH2L binding (e.g., sites similar to the β-globin LCR)

• Integrative analysis:

  • Correlate ASH2L binding with H3K4me3 ChIP-seq data

  • Integrate with RNA-seq to associate binding with gene expression

  • Compare ASH2L binding sites with known binding motifs of interacting transcription factors

  • Look for co-occupancy with other complex members (WDR5, RbBP5, MLL family proteins)

• Functional validation:

  • Select candidate binding sites for targeted ChIP-qPCR validation

  • Perform mutagenesis of binding sites (e.g., similar to the HS2mut approach)

  • Assess impact on gene expression and H3K4 methylation

  • Consider ASH2L knockdown/rescue experiments with wild-type and mutant proteins

What control experiments are essential when interpreting ASH2L antibody results?

When interpreting results from experiments using ASH2L antibodies, several control experiments are crucial:

Published studies have demonstrated the importance of these controls, showing that ASH2L knockdown in erythroid cells decreases H3K4me3 and β-globin expression, which can be rescued by wild-type ASH2L but not by DNA-binding mutants .

How can ASH2L antibodies be used to investigate its role in cancer progression?

ASH2L antibodies enable investigation of its oncogenic functions through several approaches:

• Expression analysis in clinical samples:

  • Quantify ASH2L levels in tumor versus normal tissues using immunohistochemistry

  • Correlate expression with clinical parameters and patient outcomes

  • Example: Higher ASH2L expression in endometrial cancer correlates with poor prognosis

• Functional studies in cancer models:

  • Knockdown ASH2L in cancer cell lines and assess effects on:

    • Cell proliferation and migration

    • Gene expression profiles

    • Histone modification patterns

  • Example: Depletion of ASH2L suppresses endometrial cancer cell proliferation and migration

• Mechanism exploration:

  • Investigate ASH2L-containing complexes in cancer cells using co-immunoprecipitation

  • Map genome-wide ASH2L binding in cancer versus normal cells using ChIP-seq

  • Study ASH2L's role in oncogenic signaling pathways like estrogen-ERα signaling

  • Example: ASH2L enhances ERα-mediated transactivation and regulates expression of genes like PAX2

• Therapeutic targeting assessment:

  • Monitor changes in ASH2L localization or complex formation in response to treatments

  • Evaluate compounds that disrupt ASH2L interactions or function

  • Identify biomarkers for patient stratification based on ASH2L status

What methodologies can reveal ASH2L's involvement in histone modification patterns?

Researchers can employ several techniques to study ASH2L's role in establishing histone modification patterns:

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with ASH2L antibody

  • Elute complexes and perform second immunoprecipitation with antibodies against H3K4me3

  • Analyze co-occupied regions to directly link ASH2L presence with H3K4 trimethylation

  • Example application: Demonstrating ASH2L and H3K4me3 co-occurrence at specific loci like β-globin

• Histone methyltransferase assays:

  • Immunoprecipitate ASH2L-containing complexes

  • Incubate with recombinant histone H3 and S-adenosyl methionine

  • Analyze methylation by autoradiography or methylation-specific antibodies

  • Use H3 mutants (K4A, K9A, K27A) to confirm specificity for H3K4

• Genome-wide correlation studies:

  • Perform ChIP-seq for ASH2L and various histone modifications

  • Analyze correlation between ASH2L binding and modification patterns

  • Create chromatin state maps based on histone mark combinations

  • Integrate with transcriptome data to link to gene regulation

• Domain-specific function analysis:

  • Express wild-type or mutant ASH2L in cells with depleted endogenous ASH2L

  • Assess rescue of histone modification patterns

  • Example: ASH2L DNA-binding mutants (K225E, K229E) fail to rescue H3K4me3 levels at target genes

How can researchers investigate the interplay between ASH2L and specific transcription factors?

To elucidate ASH2L's cooperation with transcription factors:

• Identification of interaction partners:

  • Perform immunoprecipitation with ASH2L antibody followed by mass spectrometry

  • Validate individual interactions by co-immunoprecipitation

  • Examples: ASH2L specifically interacts with Ap2δ and ERα

• Recruitment mechanisms:

  • Design ChIP-seq experiments for both ASH2L and the transcription factor

  • Analyze overlapping binding sites

  • Perform sequential ChIP to confirm co-occupancy

  • Use knockdown of the transcription factor to assess ASH2L recruitment dependency

  • Example: ASH2L is recruited to the β-globin locus via interaction with NF-E2

• Functional cooperation studies:

  • Establish reporter gene assays with promoters containing transcription factor binding sites

  • Co-express ASH2L and measure transcriptional enhancement

  • Use domain mutants to map regions required for functional cooperation

  • Example: ASH2L increases Ap2δ-mediated transactivation in a dose-dependent manner

• Target gene regulation:

  • Perform RNA-seq after knockdown of ASH2L, the transcription factor, or both

  • Identify cooperatively regulated genes

  • Validate selected targets by RT-qPCR and ChIP-qPCR

  • Example: ASH2L is necessary for recruitment of histone methyltransferases to Hoxc8 locus and subsequent gene activation