HEN2 Antibody

Structure and Function

HEN2, also known as NHLH2 (Nescient helix-loop-helix 2) or BHLHA34 (Class A basic helix-loop-helix protein 34), is a DNA-binding protein that contains one basic helix-loop-helix (bHLH) domain . This domain is critical for its function as a transcription factor. The protein has a calculated molecular weight of approximately 15,018 Daltons . HEN2 primarily functions in the control of cell-type determination, particularly within the developing nervous system .

Expression and Localization

HEN2 is predominantly expressed in brain tissue . At the subcellular level, HEN2 is localized to the nucleus and is found within transcription factor complexes . For efficient DNA binding, HEN2 requires dimerization with another bHLH protein, which is essential for its transcriptional regulatory functions . This dimerization capability allows HEN2 to form functional complexes with other regulatory proteins involved in neural development.

HEN2 antibodies are typically generated using synthesized peptide fragments derived from the human HEN2 protein. AFSBio's antibody utilizes an immunogen corresponding to a region within the internal amino acids of human HEN2 . Boster Bio's antibody is raised against a synthesized peptide derived from human HEN2 protein at amino acid range 30-110 . The purification methods employ affinity chromatography techniques, with AFSBio using peptide affinity chromatography with SulfoLinkTM Coupling Resin (Thermo Fisher Scientific) , while G-Biosciences purifies their antibody from rabbit antiserum using epitope-specific immunogen affinity chromatography .

Specificity and Validation

HEN2 antibodies are designed to detect endogenous levels of total HEN2 protein . Manufacturers validate their antibodies through various techniques to ensure specificity and high affinity. Boster Bio, for example, validates all antibodies on Western blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence, and ELISA with known positive control and negative samples . These validation steps are crucial for confirming antibody specificity and minimizing cross-reactivity with other proteins.

Experimental Applications and Recommended Dilutions

HEN2 antibodies are versatile tools applicable in various laboratory techniques. The primary applications include Western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). The recommended working dilutions vary depending on the specific application:

These recommended dilutions serve as starting points for assay optimization, and researchers may need to adjust dilutions based on their specific experimental conditions and sample types .

Species Reactivity

All three commercial HEN2 antibodies discussed show reactivity against both human and mouse HEN2 proteins . This cross-species reactivity makes these antibodies valuable for comparative studies between human and murine models, facilitating translational research in areas such as neurodevelopment and oncology.

Role in Neuroblastoma Development

Research has revealed that HEN2 acts as an oncogene in neuroblastoma development, cooperating with LMO3 (LIM domain only 3) to enhance the transcription of MASH1, a neuronal differentiation factor . LMO3 forms a complex with HEN2 and acts as an upstream mediator for MASH1 transcription in neuroblastoma cell lines . This oncogenic collaboration between LMO3 and HEN2 highlights the importance of HEN2 in neuroblastoma pathogenesis and identifies it as a potential therapeutic target.

Interactions with Regulatory Proteins

HEN2 interacts with multiple regulatory proteins, particularly HES1 (hairy and enhancer of split-1), a key transcriptional repressor in neuronal development. Immunoprecipitation experiments have demonstrated that HEN2 forms a complex with HES1 in cells . The interaction between HEN2 and HES1 modulates the transcriptional regulation of downstream target genes, including MASH1 .

Chromatin immunoprecipitation (ChIP) assays have shown that HEN2 can be recruited to HES1-binding sites and E-box elements in the presence of LMO3 . Interestingly, LMO3 enhances the recruitment of HEN2 to these regulatory DNA elements while simultaneously decreasing the binding of HES1 . This molecular mechanism suggests that the LMO3/HEN2 complex reduces the inhibitory effect of HES1 on MASH1 expression, thereby promoting neuronal differentiation pathways that may contribute to neuroblastoma development .

Complex Formation and Transcriptional Regulation

Research using HEN2 antibodies has revealed important insights into the mechanism of action of HEN2 in transcriptional regulation. In vitro pull-down assays and immunoprecipitation experiments have confirmed that radio-labeled FLAG-HEN2 co-immunoprecipitates with Myc-HES1, demonstrating a direct physical interaction between these proteins . Additionally, HEN2 has been shown to form stable complexes with both HES1 and LMO3, suggesting a tripartite regulatory mechanism .

Chromatin Binding and Gene Expression Modulation

ChIP assays have provided evidence that HEN2 binds to genomic regions containing putative HES1-binding sites and E-box elements . The presence of LMO3 influences this binding, with LMO3 enhancing the recruitment of HEN2 to these regulatory elements while simultaneously reducing HES1 binding . This competitive binding mechanism appears to be central to how the LMO3/HEN2 complex modulates gene expression, particularly of MASH1, which is normally repressed by HES1 .

Future Research Directions

The development of high-quality HEN2 antibodies has opened avenues for further research into the role of this protein in both normal development and disease states. Future studies may benefit from comprehensive approaches such as ChIP-seq analysis with LMO3 and HEN2 antibodies to identify the full spectrum of their transcriptional targets . Such genome-wide analyses could provide clearer answers about the regulatory networks controlled by HEN2 and its interacting partners.

Antibodies against HEN2 will continue to be essential tools for investigating the molecular pathways involved in neuroblastoma development and potential therapeutic approaches targeting the LMO3/HEN2/HES1 regulatory axis. As research progresses, improved antibodies with enhanced specificity, sensitivity, and validated applications in techniques like ChIP-seq may further advance our understanding of HEN2's functions.