ASH1L Antibody

What is ASH1L and what cellular functions does it regulate?

ASH1L is a 2,969 amino acid histone methyltransferase belonging to the SET2 subfamily. It contains three AT hook DNA-binding domains, one AWS domain, one BAH domain, one bromodomain, one PHD-type zinc finger, one post-SET domain, and one SET domain . As a histone methyltransferase, ASH1L primarily methylates lysine-4 of Histone H3, creating an epigenetic marker associated with transcriptional activation .

Studies have shown that ASH1L associates with the transcribed regions of active genes but not inactive genes, similar to RNA Polymerase II binding patterns . This association has been confirmed across multiple human and mouse cell lines, indicating a conserved function in gene expression regulation .

What is the tissue distribution and subcellular localization of ASH1L?

ASH1L is predominantly expressed in the nucleus, with highest levels found in brain, heart, and kidney tissues . In hematopoietic systems, ASH1L is expressed in all subpopulations of LSK (Lineage-SCA1+c-KIT+) progenitors and in selected mature cell subsets . This distribution pattern suggests tissue-specific roles in gene regulation and cellular differentiation.

What types of ASH1L antibodies are available for research?

Several validated antibodies targeting ASH1L have been described in the literature and are commercially available:

These antibodies recognize both human and mouse orthologs due to sequence homology . The selection of an appropriate antibody depends on the specific experimental application and the epitope accessibility in different experimental conditions.

How can I validate the specificity of ASH1L antibodies?

Antibody specificity can be validated through several approaches:

• Gene knockdown verification: Short-hairpin RNA (shRNA) directed against ASH1L can be used to knock down expression and confirm antibody specificity. Previous studies have used pSM2 plasmids containing shRNAs against human ASH1L (clone identification no. V2HS_175879) for this purpose .

• Multiple antibody comparison: Using independent antibodies targeting different epitopes of ASH1L (such as anti-ASH1L 296, 337ap, and ab4477) and comparing their detection patterns can confirm specificity .

• Immunoprecipitation followed by mass spectrometry: This approach can identify the specific protein being recognized by the antibody.

How should I perform ChIP assays using ASH1L antibodies?

For effective Chromatin Immunoprecipitation (ChIP) assays with ASH1L antibodies, follow this optimized protocol:

• Cross-link cells with 1% formaldehyde.

• For improved results, perform additional cross-linking with ethylene glycol bis(succinimidyl succinate) (34.5 mg dissolved in 5 ml DMSO per 1×10^7 cells in 45 ml PBS) for 30 minutes prior to formaldehyde treatment .

• Perform standard ChIP procedures.

• Quantify immunoprecipitated samples by real-time PCR using SYBR green dye.

• Include standards made via serial dilution of unprecipitated input samples.

• Perform all reactions in duplicate and average results from at least two independent experiments .

When designing primers for ASH1L ChIP-qPCR, focus on 5'-transcribed regions (+0.5 kb downstream from the transcription start site) where ASH1L enrichment is typically observed .

What are the optimal conditions for Western blotting with ASH1L antibodies?

ASH1L is a large protein (2,969 amino acids), which presents challenges for Western blotting. For optimal results:

• Use a low percentage (6-8%) SDS-PAGE gel to allow proper separation of high molecular weight proteins.

• Ensure complete transfer to membranes by using extended transfer times or specialized transfer methods for large proteins.

• Commercial antibodies like ASH5H03 have been validated for Western blotting applications .

• Include positive controls from tissues known to express high levels of ASH1L (brain, heart, or kidney extracts).

• Use proper negative controls, such as lysates from cells with ASH1L knockdown or knockout.

How can ASH1L antibodies be used to study epigenetic modifications in cancer models?

ASH1L has been implicated in several cancer types, making its study particularly relevant for cancer research:

• Anaplastic Thyroid Cancer (ATC): ASH1L is overexpressed in ATC relative to less aggressive differentiated thyroid cancers. ChIP-Seq using ASH1L antibodies revealed that ASH1L regulates the pro-oncogenic long noncoding RNA CCAT1, which is approximately 68-fold down-regulated in ASH1L knockout cells .

• Leukemia Models: ASH1L enhances MLL-AF9 target gene expression by binding directly to gene promoters and modifying local histone H3K36me2 levels. ChIP experiments with ASH1L antibodies can help identify target genes in leukemia progression .

• Dual ChIP-Seq Approach: Combining ChIP-Seq for ASH1L with ChIP-Seq for histone modifications (H3K4me3, H3K36me2) can identify genomic regions where ASH1L directly mediates epigenetic changes.

What are the technical considerations for immunoprecipitation with ASH1L antibodies?

When performing immunoprecipitation with ASH1L antibodies:

• Consider the large size of ASH1L protein (2,969 amino acids) when optimizing lysis conditions.

• Use antibodies specifically validated for IP applications, such as ASH5H03 .

• For co-IP experiments investigating ASH1L interaction partners, consider dual cross-linking approaches similar to those used in ChIP assays to stabilize protein complexes.

• When performing IP followed by mass spectrometry, include proper controls to distinguish between specific interactors and background proteins.

Why might I observe discrepancies between different ASH1L antibodies in ChIP experiments?

Several factors can contribute to discrepancies between antibodies:

• Epitope accessibility: Different epitopes may have varying accessibility in chromatin contexts. For example, the ab4477 antibody shows slightly different occupancy patterns compared to other ASH1L antibodies, possibly due to epitope accessibility differences at distinct genomic sites .

• Cross-reactivity: Some antibodies may cross-react with related proteins, especially other SET domain-containing methyltransferases.

• Post-translational modifications: PTMs near the epitope can affect antibody binding.

To address these issues, use multiple antibodies targeting different regions of ASH1L and validate findings across different cell types and experimental conditions.

How can I optimize ASH1L detection in tissues with low expression?

For tissues or cells with low ASH1L expression:

• Signal amplification: Consider using signal amplification methods such as tyramide signal amplification for immunohistochemistry.

• Enrichment strategies: Perform nuclear fractionation to concentrate the nuclear proteins before Western blotting.

• Sensitive detection methods: Use highly sensitive detection reagents like ECL Prime or fluorescent secondary antibodies with digital imaging.

• Increased antibody concentration: Titrate antibody concentrations to determine the optimal conditions for low-abundance detection.

How does ASH1L regulate hematopoietic stem cell function?

ASH1L plays a critical role in hematopoietic stem cell (HSC) biology:

• Studies using gene trap insertion alleles with >90% reduction in full-length ASH1L transcripts show that ASH1L deficiency leads to reduced long-term HSCs (LT-HSCs) that are nonfunctional in transplantation assays .

• ASH1L-deficient mice can accept wild-type bone marrow transplants without prior irradiation, suggesting a defect in HSC niche occupation or function .

• ChIP experiments using ASH1L antibodies can identify target genes regulated by ASH1L in HSCs versus committed progenitors to understand its role in stem cell maintenance versus differentiation.

What is the relationship between ASH1L and other histone modifying enzymes?

ASH1L functions within a complex network of histone modifiers:

• ASH1L primarily catalyzes H3K36me2 modification, which can influence other epigenetic marks and create a permissive chromatin environment for gene expression .

• ASH1L and MLL1 (another histone methyltransferase) can be co-localized at certain genomic loci, suggesting potential cooperative or complementary functions .

• Dual ChIP experiments with antibodies against ASH1L and other histone modifiers can reveal co-occupancy patterns and potential functional relationships.

How can ASH1L antibodies be used in single-cell epigenetic profiling?

As single-cell technologies advance, ASH1L antibodies can be incorporated into:

• Single-cell CUT&Tag or CUT&RUN: These techniques require highly specific antibodies for protein-DNA interaction mapping at single-cell resolution.

• Mass cytometry: Antibodies can be metal-labeled for mass cytometry to analyze ASH1L levels alongside other markers in heterogeneous cell populations.

• Super-resolution microscopy: Fluorescently labeled ASH1L antibodies can visualize subnuclear distribution patterns with nanometer precision.

When adapting ASH1L antibodies for these emerging applications, careful validation is essential to ensure specificity at lower protein amounts characteristic of single-cell analyses.

What therapeutic opportunities are suggested by ASH1L antibody research?

Research using ASH1L antibodies has revealed potential therapeutic applications:

• Leukemia treatment: ASH1L is critical for MLL-AF9-induced leukemic transformation, and its enzymatic activity is required for leukemogenesis. This suggests targeting ASH1L could be therapeutic in certain leukemias .

• Anaplastic Thyroid Cancer: ASH1L, its upstream regulator miR-200b-3p, and its downstream target CCAT1 represent potential therapeutic targets in ATC, one of the most aggressive human malignancies .

• Antibody-based therapies: While current research uses ASH1L antibodies primarily as tools, the development of cell-penetrating antibodies or antibody-drug conjugates targeting ASH1L in disease contexts represents a potential future direction.