BHLHE41 Antibody

What is BHLHE41 and what are its primary biological functions?

BHLHE41 (basic helix-loop-helix family member e41), also known as DEC2, SHARP1, or BHLHB3, is a nuclear transcriptional repressor belonging to the basic helix-loop-helix (bHLH) protein superfamily. This protein has a calculated molecular weight of approximately 51 kDa, though the observed molecular weight in experimental systems typically ranges from 48-50 kDa .

BHLHE41 is involved in the regulation of numerous physiological processes, including:

• Myogenesis and adipogenesis

• Circadian rhythm maintenance

• DNA repair mechanisms

• Mesenchymal stem cell property regulation

• Tissue-specific macrophage functions

• Lymphoid lineage physiology

At the molecular level, BHLHE41 functions primarily through homodimerization or heterodimerization with various partners, which influences its transcription factor function . It binds to DNA at consensus hexanucleotide sequences, such as E-box sequences and to a lesser degree, N-box sequences, thereby regulating gene expression.

How is BHLHE41 expression regulated in normal tissues?

BHLHE41 expression exhibits distinct tissue specificity and is regulated by multiple mechanisms:

• Tissue-specific expression pattern: Expression tends to be restricted to specific tissues, with notable expression in the nervous system, muscle tissue, and immune cells .

• Environmental regulation: Expression levels respond to environmental cues such as oxygen levels, with hypoxic conditions influencing BHLHE41 expression in many cell types .

• Biological event regulation: Expression changes during developmental processes and cellular differentiation events, particularly in B-cell development where BHLHE41 is weakly expressed in plasma cells compared to B-1a cells .

• Transcriptional control: During B-1a differentiation, Bhlhe41 expression is under the control of ARID3A, a member of the ARID superfamily of DNA binding proteins involved in chromatin-modulating complexes .

• Cytokine-mediated regulation: In T helper 2 (Th2) cells, BHLHE41 induction is particularly high during the late phase of differentiation and requires IL-4, which activates STAT6, which in turn induces GATA3 expression .

What criteria should be considered when selecting a BHLHE41 antibody for research applications?

When selecting a BHLHE41 antibody for research, consider these critical parameters:

• Target epitope: Determine whether you need an antibody targeting N-terminal, C-terminal, or internal regions. For example, some BHLHE41 antibodies specifically target the N-terminal region , which may be important for detecting specific isoforms or avoiding cross-reactivity.

• Clonality: Choose between:

  • Polyclonal antibodies: Recognize multiple epitopes (higher sensitivity)

  • Monoclonal antibodies: Recognize a single epitope (higher specificity)

• Host species: Select an appropriate host species (rabbit, mouse, etc.) that is compatible with your experimental design, especially for multi-color immunostaining experiments.

• Validated applications: Confirm the antibody has been validated for your specific application:

  • Western blot (WB): Typical dilutions range from 1:1000-1:4000

  • Immunohistochemistry (IHC): Typical dilutions range from 1:2000-1:8000

  • Immunofluorescence (IF): Typical dilutions range from 1:50-1:500

  • Other applications: Flow cytometry, ChIP, ELISA, or IP

• Reactivity: Verify cross-reactivity with your species of interest. Common reactivity includes human, mouse, and rat samples .

• Conjugation: Determine if you need unconjugated antibody or one conjugated to enzymes (HRP), fluorophores, or other detection molecules depending on your detection system .

How can I validate the specificity of a BHLHE41 antibody in my experimental system?

Validating antibody specificity is crucial for reliable results. Consider these methodological approaches:

• Positive and negative controls:

  • Use tissues/cells known to express BHLHE41 (positive controls) such as mouse testis tissue, human ovary tumor tissue, mouse brain tissue, or rat heart tissue .

  • Include tissues/cells with minimal BHLHE41 expression or knockout/knockdown models as negative controls.

• Molecular weight verification:

  • Confirm the detected band matches the expected molecular weight of BHLHE41 (48-50 kDa) .

  • Be aware that post-translational modifications may alter the observed molecular weight.

• Multiple antibody approach:

  • Use antibodies from different suppliers or those targeting different epitopes.

  • Concordance between antibodies increases confidence in specificity.

• Genetic approaches:

  • Use siRNA/shRNA knockdown of BHLHE41 to demonstrate reduced signal.

  • Use CRISPR/Cas9 knockout systems as definitive negative controls.

• Peptide competition:

  • Pre-incubate the antibody with excess immunizing peptide to block specific binding.

  • Signal reduction confirms epitope-specific binding.

What are the optimal conditions for detecting BHLHE41 by Western blot?

For optimal Western blot detection of BHLHE41, follow these methodological guidelines:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • For nuclear proteins like BHLHE41, consider nuclear extraction protocols

  • Load 20-50 μg of total protein per lane

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 48-50 kDa

  • Transfer to PVDF or nitrocellulose membranes (0.45 μm pore size)

  • Transfer at 100V for 60-90 minutes or overnight at 30V

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary anti-BHLHE41 antibody at 1:1000-1:4000 dilution

  • Incubate overnight at 4°C or 2 hours at room temperature

  • Use appropriate HRP-conjugated secondary antibody (1:5000-1:10000)

• Detection parameters:

  • Look for bands at 48-50 kDa (observed molecular weight)

  • Use mouse testis tissue as a positive control

  • Enhanced chemiluminescence (ECL) detection systems work well

• Optimization tips:

  • If signal is weak, increase antibody concentration or extend incubation time

  • For high background, increase washing steps or dilute antibody further

  • Consider signal enhancement systems for low expression samples

What are the key considerations for immunohistochemical detection of BHLHE41?

For successful IHC detection of BHLHE41, consider these methodological approaches:

• Tissue preparation and antigen retrieval:

  • Use formalin-fixed, paraffin-embedded (FFPE) or frozen sections

  • For FFPE sections, perform antigen retrieval with TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 (alternative)

  • Heat-induced epitope retrieval (HIER) using pressure cooker or microwave methods

• Antibody dilution and incubation:

  • Optimal dilution range: 1:2000-1:8000 for IHC applications

  • Incubate primary antibody overnight at 4°C or 1-2 hours at room temperature

  • Use appropriate detection system (HRP/DAB, fluorescent secondary antibodies)

• Controls and validation:

  • Human ovary tumor tissue, mouse brain tissue, and rat heart tissue serve as positive controls

  • Include negative controls (no primary antibody, isotype control)

  • Consider dual staining with cell-type markers for co-localization studies

• Signal development and analysis:

  • Optimize DAB development time (typically 1-10 minutes)

  • Nuclear localization is expected for BHLHE41

  • Quantify staining intensity using appropriate image analysis software

How can BHLHE41 antibodies be utilized in flow cytometry applications?

For flow cytometry applications with BHLHE41 antibodies, implement these methodological guidelines:

• Cell preparation:

  • Single-cell suspensions from tissues or cultured cells

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1-0.5% Triton X-100 or saponin-based buffers for intracellular staining

• Antibody considerations:

  • Use flow cytometry-validated BHLHE41 antibodies

  • Optimal dilution typically ranges from 1:10-1:50 for flow cytometry

  • Consider directly conjugated antibodies for multi-parameter analysis

• Controls and compensation:

  • Use isotype controls to determine background staining

  • Include FMO (fluorescence minus one) controls for multi-parameter analysis

  • Perform single-color controls for compensation when using multiple fluorochromes

• Data acquisition and analysis:

  • Gate on live cells using appropriate viability dyes

  • Analyze BHLHE41 expression in relevant cell populations

  • Consider co-staining with lineage markers for identifying specific cell subsets

How can I investigate BHLHE41's role in transcriptional regulation using ChIP assays?

Chromatin immunoprecipitation (ChIP) with BHLHE41 antibodies can elucidate its genomic binding sites and transcriptional regulatory functions. Follow these methodological guidelines:

• Chromatin preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

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

  • Verify sonication efficiency by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with ChIP-validated BHLHE41 antibody

  • Include appropriate controls (IgG, input DNA)

  • Wash beads thoroughly to remove non-specific binding

• DNA recovery and analysis:

  • Reverse crosslinks and purify DNA

  • Analyze enrichment by qPCR, ChIP-seq, or ChIP-chip

  • Focus on E-box and N-box consensus sequences known to bind BHLHE41

• Target validation:

  • Confirm binding at predicted target genes related to:

    • Cell cycle regulation

    • Apoptosis

    • Epithelial-to-mesenchymal transition

    • Hypoxia response

  • Validate binding sites with reporter assays or EMSA

What approaches can be used to study BHLHE41's role in cancer progression?

To investigate BHLHE41's dual roles as both tumor suppressor and oncogene , consider these methodological approaches:

• Expression analysis in clinical samples:

  • Analyze BHLHE41 expression in tumor vs. matched normal tissues

  • Correlate expression with clinical outcomes and tumor stage

  • Focus on cancer types with established BHLHE41 dysregulation, including:

    • Ovarian serous adenocarcinoma

    • Stomach adenocarcinoma

    • Thyroid cancer

    • Multiple myeloma

    • Acute myeloid leukemia

• Functional studies using gene modulation:

  • Overexpression systems to study oncogenic functions

  • Knockdown/knockout approaches to examine tumor suppressor roles

  • Assess effects on:

    • Cell proliferation and cell cycle progression

    • Apoptosis resistance

    • Migration and invasion capacity

    • Epithelial-to-mesenchymal transition markers

• Interaction studies:

  • Co-immunoprecipitation to identify cancer-relevant binding partners

  • Study interactions with known oncogenes and tumor suppressors

  • Investigate regulation by hypoxia-related factors

• Animal models:

  • Generate conditional knockout or transgenic overexpression mouse models

  • Analyze tumor development, progression, and metastasis

  • Evaluate therapeutic targeting potential

What are common issues with BHLHE41 detection and how can they be resolved?

How should BHLHE41 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody performance:

• Storage conditions:

  • Store at -20°C for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

  • Some formulations are stable for one year after shipment

• Buffer composition:

  • Typical storage buffer: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

  • Some preparations may contain 0.1% BSA as a stabilizer

• Working solution preparation:

  • Dilute in appropriate buffer immediately before use

  • For Western blot: dilute in 5% BSA or non-fat milk in TBST

  • For IHC/ICC: dilute in antibody diluent with background-reducing components

• Handling precautions:

  • Avoid contamination

  • Minimize exposure to light for fluorophore-conjugated antibodies

  • Follow safety guidelines when handling sodium azide-containing solutions

How is BHLHE41 dysregulation associated with cancer pathogenesis?

BHLHE41 exhibits complex roles in cancer development and progression:

• Dual functions:

  • Acts as a tumor suppressor in some cancers

  • Functions as an oncogene in others

  • Context-dependent effects based on tissue type and genetic background

• Genetic alterations:

  • Missense mutations

  • Gene amplifications

  • Gene fusions

  • Deletions have been identified in solid cancers

• Expression patterns:

  • Overexpression observed in ovarian serous adenocarcinoma, stomach adenocarcinoma, and thyroid cancer

  • High expression associated with poor outcomes in multiple myeloma and acute myeloid leukemia

• Mechanistic involvement:

  • Modulates transcriptional programs affecting:

    • Cell cycle control

    • Apoptosis regulation

    • Invasiveness

    • Epithelial-to-mesenchymal transition

    • Hypoxia response in tumor microenvironment

• Therapeutic implications:

  • Identified as an essential gene in Ewing sarcoma, multiple myeloma, Hodgkin lymphoma, and acute myeloid leukemia through RNAi screening

  • Potential biomarker and therapeutic target in specific cancer types