BHLH137 Antibody

What is BHLH137 and why is it significant in research?

BHLH137 is a transcription factor belonging to the basic helix-loop-helix (bHLH) family of proteins. It functions as a transcription factor similar to other bHLH family members like Mist1 (BHLHA15). The significance lies in its regulatory role in gene expression through binding to specific DNA sequences. BHLH137-like transcription factors have been identified in various organisms, including Solanum lycopersicum (tomato), where it is encoded by the LOC101248211 gene with an ORF length of 870bp . Understanding BHLH137 function contributes to broader knowledge of transcriptional regulation mechanisms in developmental and physiological processes across different species.

What are the recommended methods for detecting BHLH137 expression?

For detecting BHLH137 expression, researchers should consider a multi-modal approach. RNA-level detection can be performed using RT-qPCR with primers specific to the BHLH137 transcript sequence (XM_010324206.3 for the tomato homolog) . For protein-level detection, immunoblotting with BHLH137 antibodies is recommended, complemented by intracellular flow cytometry, which has proven effective for related bHLH family proteins as demonstrated in studies of Mist1 (BHLHA15) . When designing experiments, include appropriate controls similar to those used in studies of related bHLH proteins, where intracellular staining protocols successfully detected expression patterns in specific cell populations .

How should researchers validate the specificity of BHLH137 antibodies?

Validation of BHLH137 antibody specificity should follow a systematic approach including:

• Western blot analysis with recombinant BHLH137 protein as a positive control

• Comparison of signals between wild-type samples and BHLH137-knockout tissues

• Immunohistochemistry with parallel analysis using RNA probes (RNA-FISH) to confirm expression patterns

• Competitive binding assays with purified recombinant BHLH137 protein

• Cross-reactivity testing against closely related bHLH family members

Similar validation strategies have been successfully implemented for other bHLH transcription factors, including the Mist1 antibody validation using genetic knockout models (Cd23-Cre Bhlha15 fl/fl mice), which confirmed staining specificity through absence of signal in knockout samples .

What are the optimal conditions for immunoprecipitation with BHLH137 antibodies?

For successful immunoprecipitation of BHLH137, researchers should consider the following optimized protocol:

• Cell lysis buffer: Use a buffer containing 20mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 1mM EDTA with freshly added protease inhibitors

• Crosslinking conditions: 1% formaldehyde for 10 minutes at room temperature for ChIP applications

• Antibody binding: Incubate lysate with BHLH137 antibody (2-5μg) overnight at 4°C with gentle rotation

• Immunoprecipitation carriers: Protein A/G magnetic beads pre-blocked with BSA to reduce background

• Washing conditions: Perform 4-5 stringent washes with buffers of increasing salt concentration

Studies of related bHLH transcription factors have successfully employed similar approaches, such as the Bio-ChIP-seq methodology used for Mist1, which utilized an N-terminal biotin acceptor sequence for efficient pulldown and generated 30,212 common binding peaks across replicate experiments .

How can researchers effectively perform ChIP-seq with BHLH137 antibodies?

For effective ChIP-seq with BHLH137 antibodies, researchers should follow this methodological framework:

• Crosslinking optimization: Test different crosslinking times (8-12 minutes) with 1% formaldehyde

• Sonication parameters: Optimize to achieve DNA fragments of 200-500bp

• Antibody selection: Use ChIP-grade BHLH137 antibodies validated for immunoprecipitation

• IP controls: Include IgG control and input samples for normalization

• Sequencing depth: Aim for ≥20 million uniquely mapped reads per sample

• Peak calling parameters: Use stringent P-value cutoffs (<10^-10) as implemented for Mist1 ChIP-seq

• Motif analysis: Perform de novo motif discovery to identify BHLH137 binding consensus sequences

This approach mirrors successful ChIP-seq experiments with the related bHLH transcription factor Mist1, which identified 35,342 and 36,634 binding peaks across two independent experiments with significant overlap .

What are the recommended controls for BHLH137 antibody experiments?

When designing experiments with BHLH137 antibodies, the following controls are essential:

Similar control strategies have been employed in studies of related bHLH transcription factors, where conditional knockout mice (e.g., Cd23-Cre Bhlha15 fl/fl) provided critical negative controls for antibody validation .

How can researchers identify BHLH137 target genes and binding motifs?

To identify BHLH137 target genes and binding motifs, implement this integrated approach:

• Perform ChIP-seq with BHLH137 antibodies to identify genome-wide binding sites

• Conduct RNA-seq on BHLH137-deficient and control samples to identify differentially expressed genes

• Integrate ChIP-seq and RNA-seq data to identify direct targets (genes with both binding events and expression changes)

• Use de novo motif discovery tools (MEME, HOMER) on ChIP-seq peak sequences to determine consensus binding motifs

• Validate key target genes using ChIP-qPCR and luciferase reporter assays

This strategy parallels the successful approach used for Mist1, where Bio-ChIP-seq identified a consensus binding motif similar to that of E2A . The analysis revealed that 94% of E2A peaks overlapped with Mist1 peaks, suggesting similar binding preferences or potential heterodimer formation between related bHLH factors .

What methods can resolve contradictory results between BHLH137 antibodies from different sources?

When facing contradictory results between different BHLH137 antibodies, researchers should implement this systematic troubleshooting strategy:

• Epitope mapping: Determine the specific epitopes recognized by each antibody

• Cross-validation: Use orthogonal methods (e.g., mass spectrometry) to confirm protein identity

• Conditional knockout validation: Test antibodies on knockout tissues to confirm specificity

• Recombinant protein testing: Use purified BHLH137 protein to compare antibody recognition

• Post-translational modification analysis: Assess whether differences might be due to detection of different protein states

• Antibody sequencing: Consider sequencing antibodies to confirm their composition

Research on related bHLH transcription factors has demonstrated the importance of genetic validation, as shown in studies where Mist1 expression was specifically confirmed in plasma cells using knockout controls (Cd23-Cre Bhlha15 fl/fl mice) .

How can researchers study BHLH137 protein-protein interactions?

For comprehensive analysis of BHLH137 protein-protein interactions, implement these methodological approaches:

• Co-immunoprecipitation with BHLH137 antibodies followed by mass spectrometry

• Proximity-dependent biotin labeling (BioID or TurboID fused to BHLH137)

• Yeast two-hybrid screening with BHLH137 as bait

• FRET/BRET assays for direct interaction assessment in live cells

• GST pulldown assays with recombinant BHLH137 and candidate interactors

Studies of related bHLH transcription factors provide valuable methodological guidance. For instance, streptavidin pulldown of biotinylated Mist1 from nuclear extracts successfully identified protein interactions in LPS-differentiated cells . Additionally, research has demonstrated that some bHLH proteins, like Mist1, may form heterodimers with other family members such as E2A, as previously shown in a myoblast cell line .

How can researchers distinguish between BHLH137 and other closely related bHLH family members?

Distinguishing between closely related bHLH family members requires a strategic approach:

• Generate antibodies against non-conserved regions of BHLH137

• Validate antibody specificity against recombinant proteins of all related family members

• Implement high-resolution techniques like selected reaction monitoring (SRM) mass spectrometry

• Use isoform-specific siRNA/shRNA to confirm antibody specificity

• Compare expression patterns across tissues where different bHLH factors are differentially expressed

Research on related bHLH factors highlights the importance of specific detection methods. For instance, studies have successfully differentiated between bHLH family members by targeting unique regions, as demonstrated in the analysis of Mist1, which was specifically detected despite the presence of other bHLH proteins .

What are the best approaches for studying post-translational modifications of BHLH137?

To study post-translational modifications (PTMs) of BHLH137, researchers should implement:

• Phospho-specific antibodies: Develop antibodies against predicted phosphorylation sites

• Mass spectrometry analysis: Use enrichment strategies for specific PTMs (phosphorylation, ubiquitination, SUMOylation)

• 2D gel electrophoresis: Separate different BHLH137 isoforms based on charge and mass

• In vitro kinase assays: Identify kinases responsible for BHLH137 phosphorylation

• PTM site mutagenesis: Create point mutations at potential modification sites to assess functional impact

How should researchers design experiments to study BHLH137 dynamics in different cellular compartments?

For studying BHLH137 dynamics across cellular compartments, implement the following experimental design:

• Subcellular fractionation: Prepare nuclear, cytoplasmic, and other relevant fractions

• Immunofluorescence microscopy: Use BHLH137 antibodies with compartment-specific markers

• Live cell imaging: Generate BHLH137-fluorescent protein fusions to track localization in real-time

• Nuclear export/import inhibitors: Use compounds like leptomycin B to study trafficking

• Mutation analysis: Create mutations in predicted nuclear localization/export signals

• Stimulus-response experiments: Monitor BHLH137 translocation following relevant stimuli

Studies of related bHLH transcription factors have employed similar approaches. For instance, the subcellular localization and dynamics of Mist1 were effectively studied using intracellular staining and organelle trackers to visualize its distribution in relation to cellular compartments like the ER, Golgi, and lysosomes .

How can researchers integrate BHLH137 antibody data with other omics approaches?

For effective integration of BHLH137 antibody data with other omics approaches, implement this multi-layered strategy:

• ChIP-seq + RNA-seq integration: Identify direct BHLH137 targets by correlating binding events with expression changes

• ChIP-seq + ATAC-seq integration: Determine how BHLH137 binding affects chromatin accessibility

• Proteomics + ChIP-seq integration: Correlate BHLH137 interactions with genomic binding patterns

• Single-cell approaches: Combine scRNA-seq with antibody-based protein detection (CITE-seq)

• Multi-omics data visualization: Use integrated visualization tools to interpret complex datasets

Studies of related bHLH transcription factors demonstrate the power of this approach. For example, research on Mist1 successfully integrated Bio-ChIP-seq data with RNA-seq of Cd23-Cre Bhlha15 fl/fl and control plasma cells, revealing genes regulated by this transcription factor . This integrated analysis identified both activated and repressed genes, providing insights into the transcription factor's regulatory network.

What experimental approaches can determine if BHLH137 functions as a homodimer or heterodimer?

To determine BHLH137 dimerization properties, researchers should implement:

• Sequential ChIP (Re-ChIP): Pull down with BHLH137 antibody followed by antibodies against potential partners

• Proximity ligation assay (PLA): Use antibodies against BHLH137 and potential partners

• FRET/BRET analysis: Create fusion proteins with different fluorophores/luciferase

• Analytical ultracentrifugation: Assess recombinant BHLH137 oligomerization state

• Size exclusion chromatography: Separate different BHLH137 complex forms

• Native gel electrophoresis: Compare migration patterns with and without crosslinking

Research on related bHLH factors provides valuable precedent. Studies on Mist1 revealed potential heterodimer formation with E2A, as 94% of E2A binding peaks overlapped with Mist1 peaks . Additionally, streptavidin pulldown experiments with biotinylated Mist1 successfully identified interaction partners, demonstrating the effectiveness of this approach for studying bHLH protein interactions .