BHLH154 Antibody

What is BHLHA15 and why is it significant for research?

BHLHA15 (basic helix-loop-helix protein 15), also known as MIST1 (muscle intestine and stomach expression 1), belongs to the bHLH family of transcription factors. It plays a critical role in regulating the transcriptional activity of MYOD1 in muscle cell development and serves as a key regulator of acinar cell function. BHLHA15 contains a basic helix-loop-helix domain that enables it to bind to E-box motifs either as a homodimer or as a heterodimer with E-proteins. This protein may also negatively regulate bHLH-mediated transcription through its N-terminal domain . The significance of BHLHA15 in research stems from its involvement in cellular differentiation pathways and tissue-specific transcriptional regulation, making it an important target for developmental biology and cancer research.

What applications are suitable for BHLHA15 antibodies?

BHLHA15 antibodies have been validated for multiple research applications including enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunofluorescence (IF). Each application provides different insights into BHLHA15 expression and function. ELISA allows for quantitative detection in solution, Western blotting enables size determination and relative quantification, IHC-P reveals tissue and cellular localization patterns in preserved specimens, and IF provides high-resolution subcellular localization information . When designing experiments, researchers should consider the specific application requirements and select appropriate positive and negative controls to validate antibody performance in their experimental system.

What is the discrepancy between observed and calculated molecular weights for BHLHA15?

The observed molecular weight of BHLHA15 in experimental settings is approximately 68 kDa, while the calculated molecular weight based on amino acid sequence is 20,818 Da . This significant discrepancy can be attributed to several factors: post-translational modifications (such as phosphorylation, glycosylation, or ubiquitination), the presence of splice variants, protein-protein interactions that resist complete denaturation, or anomalous migration behavior in SDS-PAGE due to amino acid composition. When interpreting Western blot results, researchers should be aware of this discrepancy and consider additional validation methods such as mass spectrometry or immunoprecipitation followed by protein identification to confirm target specificity.

How can cross-reactivity affect experimental design when using BHLHA15 antibodies?

• Include proper negative controls (such as knockout tissues or BHLHA15-negative cell lines)

• Consider pre-adsorption tests with blocking peptides to confirm specificity

• Validate antibody specificity in each species and tissue type being studied

• Be aware that epitope accessibility may differ across species due to potential differences in protein folding or post-translational modifications

For applications requiring absolute specificity, researchers might consider using multiple antibodies targeting different epitopes or complementary approaches such as RNA-level detection methods.

What methodological considerations should be addressed when using BHLHA15 antibodies for investigating transcription factor dynamics?

Investigating transcription factor dynamics with BHLHA15 antibodies requires addressing several methodological considerations:

• Fixation and epitope accessibility: BHLHA15 is a nuclear protein, so fixation protocols must maintain nuclear architecture while ensuring epitope accessibility. Mild fixatives like 2-4% paraformaldehyde are often preferred over harsh crosslinkers.

• DNA binding interference: The immunogen for the BHLHA15 antibody (A10502) is located within amino acids 60-110 , which may overlap with functional domains. Researchers should verify that antibody binding doesn't interfere with DNA binding by using electrophoretic mobility shift assays (EMSA) in the presence and absence of antibodies.

• Protein-protein interaction studies: When using BHLHA15 antibodies in co-immunoprecipitation studies, researchers should determine whether the antibody disrupts protein-protein interactions, particularly with E-proteins or other bHLH family members.

• Temporal considerations: BHLHA15 expression and localization may vary temporally during cell differentiation or in response to stimuli. Time-course experiments with appropriate temporal resolution are essential for capturing dynamic changes.

• Quantitative analysis: For quantitative studies, researchers should establish standard curves using recombinant BHLHA15 protein and implement appropriate image analysis tools for immunofluorescence or immunohistochemistry quantification.

How can researchers distinguish between BHLHA15 homodimers and heterodimers in experimental settings?

Distinguishing between BHLHA15 homodimers and heterodimers requires specialized techniques and careful experimental design:

When interpreting results, researchers should consider that BHLHA15 can bind to E-box motifs as both a homodimer and a heterodimer with E-proteins . Controls with known homodimeric and heterodimeric protein pairs are essential for validating experimental approaches.

What controls are essential when validating a new lot of BHLHA15 antibody?

Rigorous validation of each new BHLHA15 antibody lot is critical for ensuring experimental reproducibility. Essential controls include:

• Positive tissue controls: Use tissues known to express high levels of BHLHA15, such as pancreatic acinar cells or specific muscle tissues.

• Negative controls: Include tissues from BHLHA15 knockout models or tissues known not to express the protein.

• Blocking peptide competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific signal. The blocking peptide for BHLHA15 antibody can be purchased based on the immunogen length .

• Technical controls:

  • Omission of primary antibody

  • Isotype control (rabbit IgG for A10502)

  • Concentration gradient to determine optimal antibody dilution

• Cross-validation: Compare results using alternative detection methods (e.g., mRNA expression via qPCR, or alternative antibodies targeting different epitopes).

Documentation of all validation experiments should be maintained for reference and included in materials and methods sections of publications.

How should researchers address potential discrepancies in BHLHA15 detection across different experimental platforms?

When researchers encounter discrepancies in BHLHA15 detection across different experimental platforms (e.g., Western blot vs. immunohistochemistry), a systematic troubleshooting approach is necessary:

• Epitope accessibility assessment: Different sample preparation methods may affect epitope exposure. For the BHLHA15 antibody, the immunogen is located within amino acids 60-110 , which may be differentially accessible depending on protein conformation in various applications.

• Protocol optimization matrix:

  • Test multiple antigen retrieval methods for IHC-P

  • Evaluate different lysis buffers for protein extraction

  • Optimize antibody concentration separately for each application

  • Vary incubation times and temperatures

• Confirmatory approaches: Implement orthogonal methods to verify findings:

  • RNA-level detection (RT-PCR, RNA-seq)

  • Alternative antibodies targeting different epitopes

  • Mass spectrometry-based protein identification

• Technical considerations:

  • Ensure proper protein transfer in Western blotting

  • Verify signal specificity using appropriate controls

  • Consider tissue-specific post-translational modifications

By systematically addressing these factors, researchers can identify the source of discrepancies and establish reliable detection protocols across experimental platforms.

How can researchers accurately interpret BHLHA15 expression in the context of cellular differentiation?

BHLHA15/MIST1 functions as a key regulator of acinar cell differentiation and function . When analyzing BHLHA15 expression in differentiation studies, researchers should consider:

• Temporal expression patterns: BHLHA15 expression may vary throughout differentiation. Time-course experiments capturing expression at multiple differentiation stages are essential for comprehensive analysis.

• Co-expression analysis: Analyze BHLHA15 in relation to other differentiation markers. Create co-expression maps to contextualize BHLHA15 within differentiation pathways.

• Functional correlation: Correlate BHLHA15 expression levels with functional outcomes such as secretory capacity, cell morphology, or expression of acinar-specific genes.

• Single-cell resolution: Population-level analyses may mask heterogeneity. Single-cell approaches (flow cytometry, single-cell RNA-seq, multiplexed immunofluorescence) can reveal subpopulations with distinct BHLHA15 expression patterns.

• Quantification methods: Implement rigorous quantification approaches with appropriate statistical analyses. For immunohistochemistry or immunofluorescence, consider both intensity and percentage of positive cells.

When reporting results, researchers should clearly describe the differentiation stage, provide comprehensive methods for BHLHA15 detection, and include both representative images and quantitative data.

What statistical approaches are recommended for analyzing BHLHA15 expression data across different experimental conditions?

Robust statistical analysis of BHLHA15 expression data requires thoughtful consideration of experimental design and data characteristics:

• Normalization strategies:

  • For Western blot data: Normalize BHLHA15 signal to appropriate loading controls (β-actin, GAPDH)

  • For qPCR: Apply validated reference genes stable under experimental conditions

  • For immunohistochemistry: Consider both staining intensity and percentage of positive cells

• Recommended statistical tests:

  • For comparing two groups: t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For paired observations: Paired t-test or Wilcoxon signed-rank test

  • For correlation analysis: Pearson (linear) or Spearman (non-parametric) correlation coefficients

• Replication requirements:

  • Minimum three biological replicates

  • Technical replicates to account for methodological variation

  • Power analysis to determine appropriate sample size

• Advanced approaches:

  • For complex datasets: Consider multivariate analysis, principal component analysis

  • For time-course experiments: Repeated measures ANOVA or mixed-effects models

  • For spatial data: Spatial statistics to analyze distribution patterns

Researchers should clearly report all statistical methods, including software packages, significance thresholds, and approaches to outlier identification and management.

How can emerging antibody technologies enhance BHLHA15 research applications?

Recent technological advances offer new opportunities for BHLHA15 research:

• Single-domain antibodies and nanobodies:

  • Smaller size allows better tissue penetration

  • Potentially improved access to sterically hindered epitopes

  • Compatibility with super-resolution microscopy techniques

• Recombinant antibody fragments:

  • Consistent lot-to-lot performance

  • Customizable formats (Fab, scFv)

  • Potential for site-specific conjugation of fluorophores or enzymes

• Proximity-based applications:

  • Antibody-based proximity ligation assays for detecting BHLHA15 interactions

  • Proximity-dependent biotin identification (BioID) to map BHLHA15 interaction networks

  • APEX2-based proximity labeling for ultrastructural localization

• Multiplexed detection systems:

  • Cyclic immunofluorescence for co-detection of multiple markers

  • Mass cytometry (CyTOF) for high-dimensional protein analysis

  • Multiplexed ion beam imaging (MIBI) for spatial proteomic analysis

These emerging technologies extend beyond traditional applications (ELISA, WB, IHC-P, IF) and enable researchers to address more complex questions about BHLHA15 localization, interactions, and function in diverse cellular contexts.

What specific considerations should be addressed when using BHLHA15 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) using BHLHA15 antibodies presents specific challenges requiring careful optimization:

• Antibody qualification for ChIP:

  • Verify that the antibody recognizes the native, chromatin-bound conformation

  • Confirm that the epitope (within amino acids 60-110 for A10502) remains accessible in crosslinked chromatin

  • Test multiple antibody concentrations to determine optimal enrichment

• Crosslinking optimization:

  • Titrate formaldehyde concentration (typically 0.1-1%) and crosslinking time

  • Consider dual crosslinkers for detecting indirect DNA associations

  • Include appropriate controls to assess crosslinking efficiency

• Sonication/fragmentation parameters:

  • Optimize sonication conditions to achieve 200-500 bp fragments

  • Verify fragment size distribution by gel electrophoresis

  • Consider enzymatic fragmentation alternatives for difficult-to-sonicate samples

• ChIP-specific controls:

  • Input samples to normalize for DNA quantity

  • IgG control to establish background enrichment levels

  • Positive control loci (known BHLHA15 binding sites at E-box motifs)

  • Negative control regions (genomic regions lacking E-box motifs)

• Data analysis considerations:

  • Apply appropriate normalization methods (input, spike-in, etc.)

  • Use peak calling algorithms suitable for transcription factor ChIP

  • Integrate with motif analysis to confirm enrichment at E-box sequences

When publishing ChIP results, researchers should provide comprehensive methodological details including antibody validation, crosslinking conditions, sonication parameters, and data analysis pipelines to ensure reproducibility.