BHLH41 Antibody

What is BHLHE41 and why is it important in biological research?

BHLHE41, also known by several synonyms including DEC2, SHARP1, and BHLHB3, is a Class E basic helix-loop-helix protein that functions as a transcription factor involved in various crucial cellular processes. This protein plays a significant role in regulating circadian rhythms, which are essential for maintaining proper physiological functions in organisms across a 24-hour cycle. BHLHE41 is also heavily involved in cellular differentiation processes, making it an important subject for developmental biology research. The protein acts as a key regulator of gene expression and plays a crucial role in controlling cell growth and differentiation pathways in multiple tissue types. Due to its involvement in circadian rhythms and cellular responses to environmental stimuli, BHLHE41 has become a promising target for research in chronobiology, developmental biology, and related disciplines . Recent research has also highlighted its potential importance in cancer biology, particularly in lung adenocarcinoma progression, suggesting broader implications for oncology research .

What types of BHLHE41 antibodies are available for research purposes?

Researchers investigating BHLHE41 have access to multiple antibody types optimized for different experimental approaches and model systems. The rabbit polyclonal antibody (e.g., CAB10555) represents one major category, which is produced through rabbit immunization and exhibits high reactivity with human samples. These polyclonal antibodies recognize multiple epitopes on the BHLHE41 protein, potentially increasing detection sensitivity across various applications. The immunogen for such antibodies typically includes recombinant fusion proteins containing amino acid sequences corresponding to portions of human BHLHE41, such as amino acids 120-280 of human BHLHE41 (NP_110389.1) . Alternatively, mouse monoclonal antibodies like OTI8H2 are available for more specific epitope targeting. These monoclonal antibodies are typically validated for techniques such as immunohistochemistry on paraffin-embedded tissues and Western blotting protocols. The monoclonal antibodies are often generated using human recombinant protein fragments corresponding to specific amino acid regions, such as amino acids 1-297 of human BHLHE41 produced in E. coli expression systems . Both antibody types have distinct advantages depending on the experimental context, with polyclonals offering broader epitope recognition and monoclonals providing higher specificity for particular protein domains.

What are the recommended applications for BHLHE41 antibodies?

BHLHE41 antibodies have been validated for several key research applications that allow investigators to detect and quantify this transcription factor across multiple experimental systems. Western blotting represents a primary application, with recommended dilutions typically ranging from 1:1000 to 1:2000 for polyclonal antibodies like CAB10555, enabling researchers to detect the BHLHE41 protein in cell and tissue lysates while assessing its molecular weight and expression levels . Enzyme-linked immunosorbent assay (ELISA) protocols have also been established for these antibodies, allowing for quantitative measurement of BHLHE41 protein concentrations in solution. For visualizing the protein's spatial distribution within tissues, immunohistochemistry (IHC) on paraffin-embedded sections has been validated for certain monoclonal antibodies such as OTI8H2 . Each application requires specific optimization parameters, including buffer compositions, incubation times, and detection methods that must be tailored to the particular research question. When designing experiments, researchers should consider the reactivity profile of each antibody, with some showing specificity for human samples while others may cross-react with multiple species like rat or mouse models, enabling comparative studies across different experimental organisms.

How should researchers optimize Western blot protocols for BHLHE41 detection?

When optimizing Western blot protocols for BHLHE41 detection, researchers must carefully consider several critical parameters to ensure specific and sensitive protein visualization. Sample preparation represents the first crucial step, with protocols typically involving cell lysis in buffer containing protease inhibitors to prevent degradation of the target protein during extraction. For BHLHE41 specifically, nuclear extraction protocols may yield better results since this protein functions as a transcription factor predominantly localized in the nucleus. Gel percentage selection should account for BHLHE41's molecular weight, with 10-12% SDS-PAGE gels typically providing optimal separation. When transferring to membranes, PVDF membranes often yield better results than nitrocellulose for this particular protein due to their higher protein binding capacity. Blocking solutions containing 5% non-fat dry milk or bovine serum albumin in TBST (Tris-buffered saline with 0.1% Tween-20) for 1-2 hours at room temperature are generally effective in reducing background signal . For primary antibody incubation, researchers should follow the manufacturer's recommended dilutions (e.g., 1:1000-1:2000 for the rabbit polyclonal antibody CAB10555) and incubate overnight at 4°C to maximize sensitivity while maintaining specificity . Visualization methods may vary depending on laboratory resources, with both chemiluminescence and fluorescence-based detection systems proven effective for BHLHE41 visualization.

What controls should be included when performing immunohistochemistry with BHLHE41 antibodies?

Proper experimental controls are essential when performing immunohistochemistry (IHC) with BHLHE41 antibodies to ensure result validity and facilitate accurate interpretation. Positive tissue controls should always be included, with known BHLHE41-expressing tissues such as normal lung epithelial cells serving as reliable reference standards based on previously published expression patterns . This approach allows researchers to confirm that their staining protocol effectively detects the target protein under the established conditions. Equally important are negative controls, which should include sections processed identically but with primary antibody omitted or replaced with non-specific IgG of the same isotype and concentration (e.g., rabbit IgG for polyclonal antibodies or mouse IgG1 for monoclonal antibodies like OTI8H2) . For advanced applications, researchers should consider including tissue from knockdown or knockout models where BHLHE41 expression has been experimentally reduced or eliminated, providing the most stringent control for antibody specificity. Antigen retrieval method optimization is particularly important for BHLHE41 detection in fixed tissues, with heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) being common starting points that may require empirical optimization. Signal development time should be standardized across all experimental and control samples to ensure valid comparisons of staining intensity, particularly when evaluating differential expression levels between normal and pathological tissues.

How can researchers verify the specificity of BHLHE41 antibodies?

Verifying antibody specificity is a critical step in BHLHE41 research to ensure experimental results accurately reflect the protein's true biological properties. Multiple complementary approaches should be employed to comprehensively assess specificity. Pre-absorption tests represent one important validation strategy, where the antibody is pre-incubated with excess purified BHLHE41 protein or the immunizing peptide before application to samples; a significant reduction in signal indicates that the antibody is binding specifically to the target epitope. Western blot analysis should reveal a single band (or expected pattern of bands in case of known isoforms) at the predicted molecular weight of BHLHE41, with minimal non-specific binding to other proteins . For more rigorous validation, researchers should test the antibody in cells or tissues with genetic manipulation of BHLHE41 expression—antibody signals should decrease proportionally in knockdown models and disappear in knockout models. Cross-reactivity testing across multiple species is essential when planning comparative studies, as antibodies may have different species specificities; for example, some BHLHE41 antibodies react with both human and rat samples, while others might be human-specific . Additionally, comparing results from multiple antibodies targeting different epitopes of BHLHE41 can provide further confidence in specificity, as consistent results across different antibodies strongly suggest specific detection of the target protein rather than cross-reactive artifacts.

How can BHLHE41 antibodies be used to study circadian rhythm regulation?

BHLHE41 (also known as DEC2) plays a fundamental role in circadian rhythm regulation, making antibodies against this protein valuable tools for chronobiology research. Investigators can employ BHLHE41 antibodies in chromatin immunoprecipitation (ChIP) assays to identify genomic regions directly bound by this transcription factor, revealing its regulatory targets within the circadian transcriptional network. When designing ChIP experiments, researchers should optimize crosslinking conditions (typically 1% formaldehyde for 10-15 minutes) and sonication parameters to generate chromatin fragments of approximately 200-500 bp for optimal immunoprecipitation with BHLHE41 antibodies. Time-course immunofluorescence studies using these antibodies allow visualization of BHLHE41's subcellular localization changes throughout the circadian cycle, with nuclear-cytoplasmic shuttling often correlating with its transcriptional activity states. Co-immunoprecipitation (Co-IP) experiments can identify BHLHE41's protein interaction partners within the circadian clock machinery, such as its known interactions with other clock components. For studying mutations associated with sleep phenotypes, researchers can use antibodies to compare wild-type BHLHE41 with mutant variants (such as those linked to short sleep duration) to assess differences in protein stability, localization, or interaction patterns . Importantly, when designing circadian experiments, samples should be collected at multiple time points throughout the 24-hour cycle (typically every 4-6 hours) to capture the oscillatory expression patterns characteristic of clock components.

How can researchers implement BHLHE41 antibodies in studies of cellular differentiation?

BHLHE41's established role in regulating cellular differentiation across multiple tissue types creates opportunities for antibody-based investigations into developmental processes and differentiation dynamics. Researchers can implement time-course immunostaining approaches during in vitro differentiation protocols, using BHLHE41 antibodies to track expression changes as stem or progenitor cells transition toward specialized cell types. This approach works particularly well in organoid culture systems, where three-dimensional architecture preserves tissue-like organization while allowing antibody accessibility for immunofluorescence analysis. Flow cytometry applications using permeabilization protocols compatible with transcription factor detection can enable quantitative assessment of BHLHE41 expression levels in heterogeneous cell populations undergoing differentiation, allowing correlation with other lineage-specific markers. For developmental studies, immunohistochemistry on embryonic tissues at different gestational stages can map the spatiotemporal expression pattern of BHLHE41 across organogenesis processes. When studying differentiation, co-immunoprecipitation with BHLHE41 antibodies followed by mass spectrometry can identify stage-specific protein interaction partners that may regulate its activity during lineage commitment. Chromatin immunoprecipitation sequencing (ChIP-seq) applications using validated BHLHE41 antibodies can reveal genome-wide binding patterns that change during differentiation, identifying direct transcriptional targets that execute differentiation programs. These approaches require careful optimization of fixation and permeabilization conditions to maintain both epitope accessibility and cellular architecture, particularly in three-dimensional culture systems where antibody penetration can be challenging.

What are common challenges when using BHLHE41 antibodies and how can they be addressed?

Researchers working with BHLHE41 antibodies frequently encounter several technical challenges that require systematic troubleshooting approaches. Non-specific background staining represents a common issue in immunohistochemistry and immunofluorescence applications, which can often be addressed by optimizing blocking conditions (increasing blocking reagent concentration to 5-10% or trying alternative blockers like normal serum matching the secondary antibody species) and implementing more stringent washing protocols (increasing wash duration and buffer volume). Low signal intensity despite confirmed BHLHE41 expression might indicate epitope masking, particularly in fixed tissues, necessitating evaluation of alternative antigen retrieval methods such as extended retrieval times, different buffer compositions, or enzymatic retrieval approaches for formalin-fixed samples. Batch-to-batch variability between antibody lots can introduce inconsistency in experimental results; researchers should maintain detailed records of lot numbers and consider purchasing larger quantities of validated lots for long-term projects. Nuclear proteins like BHLHE41 may require specialized extraction protocols to ensure complete solubilization, such as high-salt nuclear extraction buffers or sonication steps, particularly when performing Western blot analysis. Cross-reactivity with related proteins, especially other basic helix-loop-helix family members with structural similarity, may necessitate additional validation steps such as peptide competition assays or testing in knockout models. For particularly challenging applications, researchers might consider using multiple antibodies targeting different epitopes of BHLHE41 and looking for convergent results as confirmation of specific detection.

How should researchers approach quantitative analysis of BHLHE41 expression in tissues?

Quantitative analysis of BHLHE41 expression in tissues requires rigorous standardization and statistical approaches to generate reliable, reproducible data. When analyzing immunohistochemically stained tissue sections, researchers should implement systematic scoring systems that account for both staining intensity (typically on a 0-3 scale where 0=negative, 1=weak, 2=moderate, 3=strong) and percentage of positive cells, often combined into H-scores or Allred scores for comprehensive quantification. Digital image analysis using specialized software packages can reduce subjective bias in scoring, with algorithms capable of distinguishing nuclear staining (expected for transcription factors like BHLHE41) from cytoplasmic background. Proper tissue sampling strategies are crucial, particularly in heterogeneous tissues like tumors, where multiple representative fields (typically 5-10) should be evaluated across different regions. Normalization to housekeeping proteins becomes essential when performing quantitative Western blot analysis of BHLHE41 expression, with careful selection of loading controls appropriate for the experimental context (e.g., avoiding controls affected by the experimental conditions). When comparing BHLHE41 expression between normal and pathological tissues, matched samples from the same patient whenever possible provide the most valid comparisons by controlling for individual variation . For correlative studies linking expression levels to clinical parameters, as demonstrated in lung adenocarcinoma research, appropriate statistical methods should be applied, with threshold determination for "positive" versus "negative" expression based on biological relevance and validated against outcomes as shown in research correlating BHLHE41 expression with lung cancer prognosis .

What considerations are important when studying BHLHE41 mutations and variants?

Studying BHLHE41 mutations and variants, such as those associated with short sleep phenotypes, requires specialized approaches to detect and characterize alterations in this transcription factor. Researchers should consider whether standard antibodies can recognize mutant variants of interest, as amino acid substitutions may potentially alter epitope structures recognized by some antibodies. For novel mutations, validation experiments comparing antibody reactivity between wild-type and mutant proteins expressed in recombinant systems can establish detection reliability. When investigating naturally occurring variants like those linked to sleep duration, custom antibodies specifically designed to differentiate between wild-type and mutant forms may be necessary for studying allele-specific expression in heterozygous samples . Functional characterization of mutations often requires complementary approaches beyond antibody-based detection, including DNA-binding assays to assess how mutations affect interaction with target genes, protein stability assays to determine if variants have altered half-lives, and subcellular localization studies to identify potential differences in nuclear import or export. For clinical studies investigating mutation frequencies in patient populations, researchers should establish whether detected protein expression abnormalities correlate with genotypic changes, as demonstrated in studies examining resistance to sleep deprivation . Additionally, when examining mutation effects on protein-protein interactions, co-immunoprecipitation experiments comparing wild-type and mutant BHLHE41 can reveal differential binding to partner proteins that may explain phenotypic differences, such as the altered sleep requirements observed in individuals carrying BHLHE41 mutations.

How might BHLHE41 antibodies contribute to understanding neurobehavioral phenotypes?

BHLHE41's established association with sleep duration and resistance to sleep deprivation opens promising avenues for antibody-based investigations into neurobehavioral phenotypes. Researchers can employ immunohistochemistry with BHLHE41 antibodies to map expression patterns across brain regions involved in sleep regulation, particularly focusing on the suprachiasmatic nucleus, ventrolateral preoptic nucleus, and other sleep-wake regulatory centers. These mapping studies should include time-point analysis across the circadian cycle to capture oscillatory expression patterns that may correlate with sleep-wake transitions. For investigating the mechanisms behind the reported resistance to neurobehavioral effects of sleep deprivation in individuals with BHLHE41 mutations, researchers can design comparative studies using antibodies to assess protein expression, subcellular localization, and post-translational modifications between wild-type and mutant variants in relevant neuronal cell types . Co-localization studies combining BHLHE41 antibodies with markers for specific neurotransmitter systems (such as GABA, histamine, or orexin) can reveal the neurochemical context of its function in sleep regulation. In animal models subjected to sleep deprivation protocols, quantitative immunohistochemistry can measure adaptive changes in BHLHE41 expression that may contribute to homeostatic sleep responses. As therapeutic approaches targeting circadian rhythm disorders advance, validated antibodies will become increasingly valuable for assessing the impact of chronotherapeutics on BHLHE41 expression and function across neural circuits regulating sleep, mood, and cognitive performance.

What role might BHLHE41 play in autophagic cell death in other cancer types?

The discovery of BHLHE41's involvement in inducing autophagic cell death in non-small cell lung cancer suggests potential broader implications across multiple cancer types that warrant investigation using antibody-based approaches. Researchers can implement systematic immunohistochemical screening of tissue microarrays from various cancer types to establish expression patterns and correlations with clinicopathological features, similar to the approach that revealed associations between BHLHE41 expression and favorable prognostic indicators in lung adenocarcinoma . These screening efforts should incorporate double immunostaining with autophagy markers like LC3 to determine whether the association between BHLHE41 expression and autophagic activity extends beyond lung cancer. For mechanistic studies, inducible expression systems combined with live-cell imaging using fluorescently-tagged LC3 can visualize autophagosome formation dynamics in response to BHLHE41 expression across cell lines derived from different cancer types. Comparative ChIP-seq studies using BHLHE41 antibodies in multiple cancer cell types can identify conserved and tissue-specific transcriptional targets related to autophagy regulation, potentially revealing context-dependent mechanisms. Therapeutic implications can be explored through combinatorial studies assessing how BHLHE41 expression affects sensitivity to autophagy modulators across cancer types, building on observations that chloroquine enhanced LC3 accumulation and suppressed cell death in BHLHE41-expressing lung cancer cells . Additionally, examining the relationship between BHLHE41 expression and response to current standard-of-care therapies may uncover its potential as a biomarker for treatment stratification, particularly for therapies known to modulate autophagy as part of their mechanism of action.

How can emerging antibody technologies enhance BHLHE41 research?

Emerging antibody technologies present exciting opportunities to advance BHLHE41 research beyond the capabilities of conventional methods. Single-cell antibody-based techniques, such as mass cytometry (CyTOF) using metal-conjugated BHLHE41 antibodies, can provide unprecedented resolution of expression heterogeneity within complex tissues, revealing how BHLHE41 levels vary across cell subpopulations in contexts like tumor microenvironments or developing tissues. Proximity ligation assays (PLA) using pairs of antibodies against BHLHE41 and potential interaction partners can visualize protein-protein interactions in situ with subcellular resolution, enabling spatial mapping of BHLHE41's interaction network across different physiological and pathological contexts. For studying dynamic processes, antibody-based biosensors incorporating conformation-sensitive antibody fragments could potentially monitor real-time changes in BHLHE41 activity or conformation in living cells. Highly multiplexed imaging technologies, such as Imaging Mass Cytometry or CODEX, allow simultaneous visualization of BHLHE41 alongside dozens of other proteins, enabling comprehensive characterization of its expression in relation to cell phenotype, signaling states, and microenvironmental factors. In the therapeutic realm, the development of intrabodies (intracellular antibodies) specifically targeting BHLHE41 could provide tools for acute functional perturbation with greater specificity than genetic approaches, enabling temporal control over BHLHE41 activity. Additionally, antibody-drug conjugates directed against surface markers on cells with aberrant BHLHE41 expression patterns might eventually represent a therapeutic strategy for targeting cancer cells with specific BHLHE41-related transcriptional profiles.