BHLH131 Antibody

What is BHLH131 and why is it important in research?

BHLH131 belongs to the basic helix-loop-helix (bHLH) family of transcription factors, which represents the second largest class of transcription factors widely distributed across plants, animals, and microorganisms. The bHLH domain comprises approximately 60 amino acids with a basic amino acid region and a helix-loop-helix region. The basic region (15-20 amino acids) at the N-terminal forms a DNA-binding domain that regulates G-box and E-box binding activity, while the HLH region (40-50 amino acids) in the C-terminal domain contains two alpha helixes separated by a loop of variable length . This structure facilitates interactions with other bHLH proteins and the formation of homodimers and heterodimers, making it crucial for understanding transcriptional regulation and protein-protein interactions in cellular pathways.

How are antibodies against bHLH transcription factors typically generated?

Antibodies against bHLH transcription factors like BHLH131 are typically generated through several approaches. The most common method involves immunizing animals (often rabbits or mice) with either synthetic peptides corresponding to unique regions of the target protein or with recombinant proteins. For enhanced immunogenicity, particularly for challenging targets, researchers often employ protein conjugation techniques, such as coupling the antigen to keyhole limpet hemocyanin (KLH) . This approach promotes T cell-dependent affinity maturation of the antibody response by B cells. Following immunization, B cells from animals showing the strongest immune response are isolated for cloning of variable heavy (VH) and variable light (VL) chains, which are then co-transfected into appropriate cell lines for antibody production . Screening methods like ELISA, Western blotting, and immunohistochemistry are used to validate specificity and sensitivity.

What are the typical structural features of the BHLH131 protein that antibodies target?

Antibodies against BHLH131 typically target distinctive structural features of the protein. The bHLH domain consists of two functionally distinct regions: the basic region for DNA binding and the HLH region for dimerization. Effective antibodies may target unique epitopes within these regions or protein-specific sequences outside the conserved bHLH domain. When designing antibodies, researchers often analyze the amino acid sequence to identify regions with:

• Low sequence homology with other bHLH family members to ensure specificity

• Surface-exposed locations to increase accessibility for antibody binding

• Low structural complexity to maintain epitope recognition in various assay conditions

• Post-translational modification sites if detecting specific protein states

These structural considerations are crucial for developing antibodies that can distinguish BHLH131 from other closely related bHLH proteins that share the conserved domain structure .

How should researchers validate the specificity of BHLH131 antibodies?

Thorough validation of BHLH131 antibodies is critical for ensuring experimental reliability. A comprehensive validation approach should include:

• ELISA Testing: Perform dose-dependent binding assays using purified recombinant BHLH131 protein versus related bHLH proteins to establish specificity profiles. Results should demonstrate specific binding to BHLH131 with minimal cross-reactivity .

• Western Blot Analysis: Test the antibody against lysates from cells with known BHLH131 expression levels, including:

  • Wild-type expressing cells

  • BHLH131 overexpression systems

  • BHLH131 knockdown/knockout models
    The antibody should detect bands of the expected molecular weight (~45-60 kDa depending on post-translational modifications) only in samples containing BHLH131 .

• Immunoprecipitation: Verify that the antibody can specifically pull down BHLH131 from complex protein mixtures, with subsequent mass spectrometry confirmation.

• Immunohistochemistry Controls: Include positive tissues known to express BHLH131 and negative controls where the protein is absent, similar to validation approaches used for other transcription factor antibodies .

• Genetic Validation: Test antibody reactivity in BHLH131-null backgrounds created through CRISPR/Cas9 or similar gene editing technologies to confirm signal absence.

This multi-platform validation approach minimizes the risk of experimental artifacts and ensures that observed signals genuinely represent BHLH131 detection .

What are the optimal protocols for using BHLH131 antibodies in Western blotting?

For optimal Western blot results with BHLH131 antibodies, researchers should consider the following protocol adaptations:

• Sample Preparation:

  • Use nuclear extraction protocols optimized for transcription factors, as BHLH131 is predominantly localized in the nucleus

  • Include protease inhibitors and phosphatase inhibitors to preserve post-translational modifications

  • Maintain samples at 4°C throughout processing to prevent degradation

• Gel Separation:

  • Use 10-12% SDS-PAGE gels for optimal separation of BHLH131 (expected MW ~45-60 kDa)

  • Include positive control lysates from cells with confirmed BHLH131 expression

• Transfer Conditions:

  • Semi-dry transfer: 15V for 30-45 minutes

  • Wet transfer: 30V overnight at 4°C for maximum protein transfer efficiency

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk or 3-5% BSA in TBS-T for 1 hour at room temperature

  • Dilute primary BHLH131 antibody 1:500 to 1:2000 (optimize for each lot)

  • Incubate overnight at 4°C with gentle rocking

• Detection and Troubleshooting:

  • Use ECL detection systems with exposure times starting at 30 seconds

  • For weak signals, consider enhanced chemiluminescence substrates or signal amplification systems

  • If background is high, increase washing times and detergent concentration in wash buffer

This methodological approach has been shown to produce reliable results for other bHLH transcription factor antibodies and should be applicable to BHLH131 detection .

How can researchers optimize immunoprecipitation protocols for BHLH131 protein complexes?

Optimizing immunoprecipitation (IP) for BHLH131 and its interacting partners requires careful consideration of protein complex preservation:

• Cell Lysis Conditions:

  • Use gentle lysis buffers (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) with protease/phosphatase inhibitors

  • For nuclear proteins like BHLH131, include a nuclear extraction step before IP

  • Sonicate minimally to avoid disrupting protein-protein interactions

• Antibody Coupling:

  • Pre-couple 2-5 μg of BHLH131 antibody to protein A/G beads or magnetic beads

  • For co-IP studies, consider cross-linking the antibody to beads using bis(sulfosuccinimidyl)suberate (BS3) to prevent antibody contamination in the eluted sample

• Binding and Washing Conditions:

  • Incubate lysate with antibody-coupled beads for 3-4 hours at 4°C with gentle rotation

  • Perform at least 4 washes with buffer containing reduced detergent concentration

  • Include salt gradient washes (150-300 mM NaCl) to reduce non-specific binding

• Elution Strategies:

  • For routine IP, use SDS sample buffer at 95°C for 5 minutes

  • For co-IP mass spectrometry analysis, consider native elution with competing peptides or low pH glycine buffers (pH 2.5-3.0) followed by immediate neutralization

This optimized approach enables researchers to capture BHLH131 along with its interacting partners, including other bHLH proteins with which it forms heterodimers, facilitating the study of transcriptional regulatory complexes .

How can BHLH131 antibodies be used to study protein-protein interactions within the bHLH family?

BHLH131 antibodies provide powerful tools for investigating complex protein-protein interactions within the bHLH transcription factor family. Advanced experimental approaches include:

• Sequential Chromatin Immunoprecipitation (ChIP-reChIP):

  • Perform primary ChIP with BHLH131 antibody

  • Elute complexes under native conditions

  • Perform secondary ChIP with antibodies against suspected partner proteins

  • This technique identifies genomic loci bound by BHLH131-containing heterodimers

• Proximity-Dependent Biotin Identification (BioID) or APEX2 Proximity Labeling:

  • Generate BHLH131-BioID or BHLH131-APEX2 fusion proteins

  • Use BHLH131 antibodies to confirm expression and localization of fusion proteins

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Förster Resonance Energy Transfer (FRET) Analysis:

  • Employ BHLH131 antibodies to validate expression levels of fluorescently tagged BHLH131 and potential partner proteins

  • Measure energy transfer between fluorophores to confirm direct protein interactions

  • Analyze interaction kinetics and affinity

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fragments are fused to BHLH131 and potential interactors

  • BHLH131 antibodies verify expression levels before visualization

  • Reconstituted fluorescence indicates interaction in living cells

These advanced techniques allow researchers to map intricate interaction networks of BHLH131 with other transcription factors, co-activators, or co-repressors, illuminating its role in transcriptional regulation .

What methods are recommended for studying BHLH131 subcellular localization across different cell types and conditions?

Investigating BHLH131 subcellular localization dynamics across different cellular contexts requires multiple complementary approaches:

• Immunofluorescence Microscopy Optimization:

  • Fixation: Compare paraformaldehyde (4%) versus methanol fixation for optimal epitope preservation

  • Permeabilization: Test Triton X-100 (0.1-0.5%) versus saponin (0.1-0.3%) for nuclear transcription factor access

  • Antibody dilution: Typically start at 1:100-1:500 for primary antibodies

  • Include co-staining with nuclear markers (DAPI), and other cellular compartment markers

• Biochemical Fractionation Coupled with Western Blotting:

  • Separate nuclear, cytoplasmic, chromatin-bound, and soluble nuclear fractions

  • Analyze BHLH131 distribution across fractions using validated antibodies

  • Include fraction-specific control proteins (e.g., Lamin B1 for nuclear envelope, GAPDH for cytoplasm)

• Live-Cell Imaging with Fluorescent Protein Tags:

  • Validate constructs using BHLH131 antibodies to confirm that tagged proteins behave like endogenous BHLH131

  • Monitor dynamic changes in localization following stimuli or during cell cycle progression

  • Combine with photobleaching techniques (FRAP) to measure mobility and binding dynamics

• Super-Resolution Microscopy:

  • Employ STORM or PALM techniques with BHLH131 antibodies for nanoscale localization

  • Analyze co-localization with chromatin marks or other transcription factors at enhanced resolution

These approaches collectively provide a comprehensive view of BHLH131 localization patterns, revealing how its distribution correlates with function across different cellular states and environmental conditions .

How can BHLH131 antibodies be applied in ChIP-seq experiments to map genome-wide binding sites?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using BHLH131 antibodies requires careful optimization to generate high-quality, reproducible binding profiles:

• Antibody Validation for ChIP Applications:

  • Perform ChIP-qPCR on known or predicted BHLH131 binding sites

  • Compare enrichment using different antibody concentrations (2-10 μg per reaction)

  • Include IgG control and input normalization

  • Verify specificity using cells with BHLH131 knockdown or knockout

• Crosslinking and Chromatin Preparation Optimization:

  • Test multiple formaldehyde concentrations (0.5-1.5%) and crosslinking times (5-20 minutes)

  • Optimize sonication conditions to achieve 200-500 bp fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Consider dual crosslinking (formaldehyde plus disuccinimidyl glutarate) for improved efficiency

• ChIP Protocol Refinements:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Incubate chromatin and antibody overnight at 4°C with rotation

  • Include extensive washing steps with increasing salt concentrations

  • Reverse crosslinks at 65°C for 4-16 hours

• Data Analysis and Validation:

  • Identify enriched regions using peak-calling algorithms like MACS2

  • Perform motif analysis to identify the consensus binding sequence for BHLH131

  • Validate novel binding sites with ChIP-qPCR

  • Correlate binding sites with gene expression data

This methodical approach enables accurate mapping of BHLH131 binding sites genome-wide, revealing its regulatory targets and potential co-regulatory networks with other transcription factors .

How should researchers interpret contradictory results between different BHLH131 antibody-based assays?

When faced with contradictory results between different antibody-based assays for BHLH131, researchers should systematically analyze potential sources of discrepancy:

• Epitope Accessibility Assessment:

  • Different assays expose different epitopes

  • Western blotting detects denatured epitopes

  • IP and ChIP require native epitope recognition

  • IF may require partially denatured structures

  Resolution Strategy: Map the epitope recognized by each antibody and select the appropriate antibody for each application based on epitope characteristics.

• Post-Translational Modification Interference:

  • Phosphorylation, acetylation, or ubiquitination may mask epitopes

  • Different cellular conditions alter BHLH131 modification states

  Resolution Strategy: Use phosphatase treatment or other relevant enzymes to remove modifications before detection. Consider using modification-specific antibodies if available.

• Isoform-Specific Detection:

  • Alternative splicing may generate BHLH131 variants

  • Different antibodies may recognize different isoforms

  Resolution Strategy: Sequence the region recognized by each antibody and compare with known isoforms. Validate with recombinant isoforms if possible.

• Experimental Condition Variables:

  Assay Type	Potential Variables	Optimization Approach
  Western Blot	Reducing conditions, buffer pH	Test non-reducing conditions, adjust buffer composition
  IP	Salt concentration, detergent type	Titrate salt (150-500 mM), try different detergents (NP-40, Triton, CHAPS)
  ChIP	Crosslinking time, sonication	Compare various crosslinking times, optimize sonication
  IHC/IF	Fixation method, antigen retrieval	Test multiple fixatives, try heat-mediated or enzymatic retrieval methods

Cross-validation using complementary techniques, such as mass spectrometry or RNA interference, can help resolve contradictions and establish which results most accurately reflect BHLH131 biology .

What are the most common artifacts in BHLH131 immunohistochemistry and how can they be avoided?

Immunohistochemistry (IHC) with BHLH131 antibodies can produce several common artifacts that require specific mitigation strategies:

• Non-specific Nuclear Staining:

  • Cause: Many bHLH antibodies can cross-react with similar transcription factors

  • Solution: Pre-absorb antibody with recombinant related bHLH proteins; include knockout/negative tissue controls; use monoclonal antibodies with demonstrated specificity against unique BHLH131 epitopes

• False-Negative Results in Nuclear Proteins:

  • Cause: Inadequate nuclear antigen retrieval or epitope masking

  • Solution: Optimize antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0); test multiple fixation times; implement amplification systems for weak signals

• Edge Artifacts and Uneven Staining:

  • Cause: Tissue drying during processing or uneven antibody distribution

  • Solution: Maintain humidity during incubations; use sufficient antibody volume; employ automated staining platforms if available

• Background Staining in Extracellular Matrix:

  • Cause: Antibody cross-reaction with extracellular matrix proteins

  • Solution: Increase blocking time (1-2 hours); use appropriate blocker (5% BSA or 10% normal serum); include additional washing steps with higher detergent concentration (0.1-0.3% Tween 20)

• Inconsistent Results Between Batches:

  • Cause: Antibody lot variation or tissue processing differences

  • Solution: Include standard positive control tissues in each run; maintain detailed protocols; consider using automated systems for consistency

These optimization strategies can significantly reduce artifacts in BHLH131 IHC staining, leading to more reliable interpretation of expression patterns across different tissue types .

How can researchers quantitatively analyze BHLH131 expression levels across different experimental conditions?

Accurate quantitative analysis of BHLH131 expression across experimental conditions requires rigorous methodological approaches:

• Western Blot Quantification:

  • Use gradient loading of samples to ensure detection in the linear range

  • Include recombinant BHLH131 protein standards for absolute quantification

  • Normalize to appropriate loading controls (nuclear proteins like Lamin B for nuclear transcription factors)

  • Employ fluorescent secondary antibodies for wider linear detection range

  • Use image analysis software with background subtraction and lane profile analysis

• Quantitative Immunofluorescence:

  • Collect images with identical exposure settings across all samples

  • Include fluorescence calibration standards in each experiment

  • Perform nuclear segmentation based on DAPI staining

  • Measure mean nuclear intensity of BHLH131 signal

  • Apply appropriate statistical tests for comparing conditions

• Flow Cytometry for BHLH131 Detection:

  • Optimize fixation and permeabilization for nuclear transcription factors

  • Include fluorescence-minus-one (FMO) controls

  • Measure median fluorescence intensity (MFI) for population analysis

  • Gate on specific cell populations when analyzing heterogeneous samples

• Quantitative Analysis Workflow:

  Analysis Step	Recommended Approach	Common Pitfalls to Avoid
  Background Correction	Use rolling ball algorithm	Manual background selection
  Normalization	Use spike-in controls	Relying solely on housekeeping genes
  Statistical Analysis	Apply non-parametric tests for small sample sizes	Using parametric tests without checking data distribution
  Data Presentation	Include all data points with mean/median indicators	Showing only fold changes without absolute values

These quantitative approaches provide robust measurements of BHLH131 expression levels, enabling meaningful comparisons across different experimental conditions and treatments .

How might BHLH131 antibodies be applied in single-cell protein analysis technologies?

Emerging single-cell protein analysis technologies offer exciting opportunities for applying BHLH131 antibodies to understand heterogeneity in transcription factor expression and activity:

• Mass Cytometry (CyTOF) Applications:

  • Metal-conjugated BHLH131 antibodies enable simultaneous detection with dozens of other proteins

  • Correlation of BHLH131 expression with cell cycle markers, signaling pathways, and other transcription factors

  • Identification of rare cell populations with unique BHLH131 expression patterns

  • Required validation: Titration experiments to determine optimal concentration of metal-conjugated antibodies

• Single-Cell Western Blot Adaptation:

  • Microfluidic platforms for analyzing BHLH131 in thousands of individual cells

  • Correlation of expression levels with functional outcomes at single-cell resolution

  • Technical considerations: Optimization of cell lysis conditions to preserve nuclear proteins

• Proximity Ligation Assay (PLA) for Protein Interactions:

  • Detection of BHLH131 interaction with specific partners in individual cells

  • Visualization of spatial distribution of interactions within subcellular compartments

  • Quantification of interaction frequency across cell populations

• Emerging Technologies:

  Technology	Application with BHLH131 Antibodies	Technical Requirements
  Imaging Mass Cytometry	Spatial mapping of BHLH131 in tissue context	Metal-conjugated primary or secondary antibodies
  Digital Spatial Profiling	Quantitative spatial analysis of BHLH131	Oligonucleotide-tagged antibodies
  SeqFISH/MERFISH	Combined protein-RNA detection at single-cell level	Optimized multiplexed immunofluorescence protocols

These technologies promise to reveal unprecedented insights into the heterogeneous expression and function of BHLH131 across different cell types and states, potentially uncovering new regulatory mechanisms and cellular functions .

What developments in antibody engineering might improve BHLH131 detection sensitivity and specificity?

Recent advances in antibody engineering offer promising approaches to enhance BHLH131 detection:

• Recombinant Antibody Fragments:

  • Single-chain variable fragments (scFvs) and antigen-binding fragments (Fabs) provide superior tissue penetration

  • Smaller size allows access to sterically hindered epitopes in protein complexes

  • Bacterial or yeast display libraries can be screened for fragments with enhanced affinity and specificity

  • Potential for site-specific labeling with fluorophores or other detection moieties

• Bi-specific Antibody Development:

  • Recognition of two distinct epitopes on BHLH131 or simultaneous binding of BHLH131 and interacting partners

  • Increased specificity through dual epitope recognition

  • Enhanced sensitivity through avidity effects

  • Applications in super-resolution microscopy and proximity-dependent detection methods

• Nanobody Technology:

  • Single-domain antibodies derived from camelid heavy-chain-only antibodies

  • Exceptional stability under various buffer conditions

  • Small size (~15 kDa) for accessing restricted epitopes

  • Potential for intracellular expression as "intrabodies" for live-cell applications

• Synthetic Biology Approaches:

  • Directed evolution of antibody binding domains using yeast or phage display

  • Computational design of binding interfaces with enhanced specificity for BHLH131

  • Integration with split reporter systems for real-time detection of BHLH131 activity

These emerging antibody technologies could significantly advance our ability to detect and monitor BHLH131 with improved sensitivity, specificity, and spatiotemporal resolution, enabling new insights into its biological functions and regulatory mechanisms .