HEY2 Antibody

What is HEY2 and why is it important in research?

HEY2 (hairy/enhancer-of-split related with YRPW motif protein 2) is a bHLH transcription factor that functions as a downstream effector of Notch signaling. The canonical human HEY2 protein consists of 337 amino acid residues with a molecular weight of approximately 35.8 kDa and localizes to the nucleus. It plays critical roles in cardiovascular development and is widely expressed across multiple tissue types. HEY2 is particularly valuable as a marker for identifying pulmonary artery smooth muscle cells and blood vessel smooth muscle cells, making it an important research target for developmental biology and cardiovascular disease studies .

What alternative names for HEY2 should researchers be aware of when searching literature?

When conducting literature searches or ordering antibodies, researchers should be aware of multiple synonyms for HEY2, including: GRIDLOCK, GRL, HERP1, HESR2, HRT2, bHLHb32, CHF1, and hairy/enhancer-of-split related with YRPW motif protein 2. Using these alternative names in database searches will ensure comprehensive literature coverage and prevent missing relevant research .

Which species have HEY2 orthologs that can be detected with commercial antibodies?

HEY2 gene orthologs have been reported and can be detected in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken. Many commercial antibodies offer cross-reactivity across these species, but researchers should verify specific reactivity profiles when selecting antibodies for non-human models. The most commonly available antibodies show reactivity to human, mouse, and rat HEY2, though specific epitope conservation should be verified depending on the experimental model .

What are the optimal experimental applications for HEY2 antibodies?

HEY2 antibodies are primarily utilized for Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunoprecipitation (IP). Based on the available products, Western Blotting appears to be the most widely validated application, with most antibodies supporting this technique. For immunolocalization studies, IF and ICC applications are well-supported by several validated antibodies. When selecting antibodies for specialized techniques such as ChIP (Chromatin Immunoprecipitation), researchers should specifically seek antibodies validated for these applications, as HEY2 directly binds to promoter regions of target genes .

What is the recommended methodology for detecting HEY2 binding to target promoters?

Chromatin Immunoprecipitation (ChIP) is the gold standard technique for detecting HEY2 binding to target promoters. Research has shown that HEY proteins directly bind to the proximal promoter regions (within 2 kb of the transcriptional start site) of target genes. When performing ChIP for HEY2, researchers should:

• Focus on proximal promoter regions (within 2 kb of TSS)

• Include negative controls with non-induced cells or IgG antibodies

• Test conserved sequences from established targets (HEY1, KLF10, BMP2, FOXC1)

• Consider ChIP-seq for genome-wide binding site identification

Research has demonstrated that sequences from proximal promoter regions can be enriched 10-60 fold in properly executed ChIP experiments, while conserved sequences further upstream (-1.4 to -6.5 kb from TSS) or intronic regions typically show little to no enrichment .

How should researchers validate HEY2 target genes identified in cell culture models?

Validation of HEY2 target genes initially identified in cell culture models should follow a multi-step process:

• Confirm binding sites through ChIP-qPCR in the model cell line

• Validate binding in physiologically relevant cell types (e.g., HL-1 cardiomyocytes for cardiac targets)

• Assess functional regulation through qRT-PCR after HEY2 induction/repression

• Examine target gene expression in Hey2 knockout mouse models

• Compare results with published ChIP-seq datasets

Research has demonstrated successful validation of HEY2 binding sites between HEK293 cells and HL-1 cardiomyocytes, with 16 out of 18 binding sites confirmed across these models. For in vivo validation, ventricular tissue from Hey2-/- embryos at E14 can provide valuable confirmation of physiological regulation .

How do HEY1, HEY2, and HEYL differ in their DNA binding and target gene regulation?

• Variations in protein stability and half-life

• Differential potency of the bHLH domains

• Cell-intrinsic mechanisms affecting cooperativity with other factors

• Contextual differences in cofactor recruitment

In HEK293 cells with inducible expression systems, HEY2 regulated a larger number of target genes and demonstrated a greater magnitude of regulation compared to HEY1, though these differences partially equalized with longer induction periods. For repressed genes, the impact was generally stronger (2-6 fold changes) compared to activated genes .

What amino acid residues are critical for HEY2 DNA binding activity?

The basic domain of HEY2, like other bHLH proteins, contains critical amino acid residues that make direct contact with DNA. Research on HEY1 has identified three arginine residues (positions equivalent to R50, R54, and R62) that are essential for DNA binding and transcriptional activity. Conservative mutations of these residues to lysine (R50K, R54K, R62K) completely abolished DNA binding ability in ChIP experiments and eliminated transcriptional repression activity.

Given the structural conservation between HEY family members, these findings likely apply to HEY2 as well. When designing experiments with mutated HEY2 constructs, researchers should consider:

• Conservative mutations that maintain protein stability and folding

• Verification of nuclear localization of mutant proteins

• Confirmation of dimerization ability

• Assessment of DNA binding through ChIP

• Functional validation through target gene expression analysis

What is the recommended approach for genome-wide identification of HEY2 binding sites?

For genome-wide identification of HEY2 binding sites, ChIP-seq (Chromatin Immunoprecipitation followed by next-generation sequencing) is the optimal methodology. Based on published protocols:

• Use tightly controlled expression systems with appropriate negative controls

• Employ high-affinity antibodies, preferably against epitope tags (Flag, HA) if using overexpression systems

• Generate 13-14 million reads minimum for comprehensive coverage

• Apply stringent peak calling criteria (p-value <10^-5 and peak height ≥10)

• Validate selected peaks through independent ChIP-qPCR

This approach has successfully identified approximately 10,000 high-confidence binding sites for both HEY1 and HEY2. Validation studies have confirmed the reliability of these identified sites, with qPCR verification showing consistent results across peaks with heights ranging from 11 to 380 .

What are common challenges when using HEY2 antibodies for Western blotting?

When using HEY2 antibodies for Western blotting, researchers frequently encounter several challenges:

• Detection of endogenous HEY2: Due to tissue-specific and sometimes low expression levels, detection of endogenous HEY2 can be difficult. Consider enriching nuclear fractions and using sensitive detection systems.

• Specificity concerns: HEY2 shares sequence similarity with other HEY family members (HEY1, HEYL), potentially leading to cross-reactivity. Verify antibody specificity using knockout/knockdown controls or overexpression systems.

• Molecular weight confirmation: While the canonical HEY2 protein has a predicted mass of 35.8 kDa, post-translational modifications may cause mobility shifts. Researchers should validate observed bands using appropriate positive controls.

• Sample preparation: As a nuclear protein, HEY2 requires proper nuclear extraction techniques. Incomplete extraction may result in false negatives even with functioning antibodies .

What controls should be included when validating HEY2 antibodies for research applications?

Comprehensive validation of HEY2 antibodies should include the following controls:

• Positive controls: Cell lines with known HEY2 expression (HEK293 cells express endogenous HEY genes) or tissues with documented HEY2 expression (cardiac tissues, particularly ventricular cardiomyocytes).

• Negative controls:

  • Primary antibody omission

  • Isotype-matched IgG controls

  • HEY2 knockout/knockdown samples when available

  • Non-induced cells in inducible expression systems

• Specificity controls: Testing for cross-reactivity with other HEY family members (HEY1, HEYL) using overexpression systems.

• Application-specific controls: For ChIP experiments, include non-binding regions (regions 1.4-6.5kb upstream or intronic regions) as negative controls .

How can HEY2 antibodies be used to study cardiovascular development and disease?

HEY2 antibodies are valuable tools for investigating cardiovascular development and disease due to HEY2's critical role in these processes. Methodological approaches include:

• Developmental studies: Immunohistochemistry and immunofluorescence to track HEY2 expression patterns during heart development, particularly in ventricular cardiomyocytes where HEY2 is the predominant HEY family member expressed.

• Congenital heart defect models: Analysis of HEY2 expression and localization in models of ventricular septal defects (VSDs) and valve defects, which are observed in Hey2 knockout mice.

• Cardiomyopathy research: Examination of HEY2 binding to target genes in models of cardiomyopathy, as Hey2 knockout hearts show evidence of ventricular cardiomyopathy.

• Notch signaling studies: Investigation of HEY2 as a downstream effector of Notch signaling in cardiovascular contexts, using antibodies to track the relationship between Notch activation and HEY2 expression.

• Lineage specificity: Utilization of HEY2 antibodies to identify and isolate pulmonary artery smooth muscle cells and blood vessel smooth muscle cells for developmental and pathophysiological studies .

What are the important considerations when studying HEY2 in knockout mouse models?

When studying HEY2 in knockout mouse models, researchers should consider several important factors:

• Compensatory mechanisms: Hey1 and HeyL may partially compensate for Hey2 loss. Consider analyzing all three HEY family members in knockout models.

• Developmental timing: Hey2 knockout effects should be examined at appropriate developmental stages. Cardiovascular phenotypes have been documented at E14 and beyond.

• Tissue specificity: Focus on ventricular cardiomyocytes, which express only Hey2 but not Hey1 or HeyL, making this tissue particularly sensitive to Hey2 knockout.

• Phenotypic variability: Anticipate and document variability in membranous VSDs, valve defects, and cardiomyopathy symptoms across individual knockout animals.

• Target gene validation: Use Hey2 knockout tissues to validate putative target genes identified in vitro, comparing expression levels between wild-type and knockout samples .