BHLH113 Antibody

What is BHLH113 and why is it important in research?

BHLH113 belongs to the basic helix-loop-helix (bHLH) family of transcription factors, which are involved in regulating gene expression through DNA binding. These proteins contain a basic DNA binding domain adjacent to two α-helices separated by a variable loop region, forming the helix-loop-helix structure. The bHLH family plays crucial roles in various biological processes including cellular differentiation, tissue development, and stress responses . BHLH113, specifically, contributes to transcriptional regulation networks, similar to other bHLH proteins like BHLH11 that participate in iron homeostasis pathways .

Research on BHLH113 is important because understanding its function, interactions, and regulation can provide insights into fundamental biological processes. Similar to other bHLH proteins that have been studied, BHLH113 likely functions within protein complexes and regulatory networks that control gene expression in response to various signals. Studying BHLH113 requires specific and validated antibodies that can precisely detect this protein in various experimental contexts.

What experimental applications are appropriate for BHLH113 antibodies?

BHLH113 antibodies can be employed in multiple experimental applications similar to those used for other bHLH family members. Based on characterization of related bHLH antibodies, the most common applications include:

• Western blotting: For detecting BHLH113 protein in cell or tissue lysates and determining its molecular weight and expression levels. The recommended concentration range for mouse monoclonal antibodies in western blotting is typically 0.2-0.5 μg/ml .

• Immunoprecipitation (IP): For isolating BHLH113 and its interacting partners from complex protein mixtures. This technique is valuable for studying protein-protein interactions as demonstrated in studies of other bHLH proteins .

• Chromatin immunoprecipitation (ChIP): For identifying genomic regions bound by BHLH113, which helps determine its target genes and regulatory functions .

• Immunofluorescence and immunocytochemistry: For visualizing the subcellular localization of BHLH113, similar to studies that have examined the nuclear and cytoplasmic distribution of other bHLH proteins .

• Microarray applications: For high-throughput screening of BHLH113 binding partners or targets .

Each application requires specific optimization of antibody concentration, sample preparation, and experimental conditions to ensure reliable results.

How should BHLH113 antibodies be stored and handled for optimal performance?

Proper storage and handling of BHLH113 antibodies are critical for maintaining their activity and specificity over time. Based on established protocols for similar antibodies, the following recommendations apply:

When working with concentrated or bioreactor-produced antibodies, adding an equal volume of glycerol as a cryoprotectant before freezing can help maintain antibody integrity during storage . When thawing frozen antibodies, allow them to thaw completely at room temperature or 4°C rather than using heat, and mix gently to ensure homogeneity.

Avoid exposure to direct light, especially for fluorophore-conjugated antibodies, and minimize contamination by using sterile technique when handling antibody solutions. Always centrifuge the antibody solution briefly before opening to collect any liquid that might be in the cap or on the walls of the tube.

How can BHLH113 antibodies be validated for specificity in research applications?

Validation of BHLH113 antibodies for specificity is a critical step before using them in research applications. Following the consortium recommendations for antibody validation in biomarker discovery , a tiered approach can be implemented:

Tier 1: Initial validation should include western blot analysis using positive and negative control samples. For BHLH113, this could involve tissues or cell lines known to express or not express the protein. The antibody should detect a single band at the expected molecular weight in positive controls and show no signal in negative controls .

Tier 2: More rigorous validation should involve multiple orthogonal techniques:

• Genetic knockdown/knockout validation: Compare antibody reactivity in wild-type versus BHLH113 knockout or knockdown samples. Reduction or elimination of signal confirms specificity .

• Recombinant protein analysis: Test the antibody against purified recombinant BHLH113 protein and similar bHLH family members to assess cross-reactivity.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is pulling down BHLH113 rather than other proteins.

• Peptide competition assay: Pre-incubation of the antibody with a peptide containing the epitope should block specific binding in subsequent applications .

Tier 3: For antibodies intended for clinical biomarker use, additional validation steps would be required, including multi-site reproducibility testing and comparison with established gold standards .

Documentation of all validation steps is essential, including specific protocols, concentrations, and experimental conditions used. This ensures reproducibility and builds confidence in the antibody's reliability.

How can researchers determine the optimal concentration of BHLH113 antibody for different experimental applications?

Determining the optimal antibody concentration requires systematic titration for each specific application. This process should be conducted with appropriate positive and negative controls to ensure both sensitivity and specificity. For BHLH113 antibodies, consider these application-specific guidelines:

For immunohistochemistry (IHC), immunofluorescence (IF), and immunocytochemistry (ICC): Start with a concentration range of 2-5 μg/ml when using mouse monoclonal antibodies . Prepare a dilution series (e.g., 1, 2, 5, and 10 μg/ml) and test against known positive controls. The optimal concentration provides strong specific staining with minimal background.

For western blotting: Begin with a concentration range of 0.2-0.5 μg/ml for mouse antibodies . Test several concentrations and select the lowest concentration that provides a clear, specific signal. Including a loading control and molecular weight markers is essential for proper interpretation.

For chromatin immunoprecipitation (ChIP): Optimal antibody amounts typically range from 2-10 μg per ChIP reaction, but this should be determined empirically. Test different antibody amounts while keeping chromatin concentration constant.

For immunoprecipitation: Start with 1-5 μg of antibody per 500 μg of protein lysate and adjust based on results.

When switching between sample types (e.g., different cell lines or tissues) or detection methods, re-optimization may be necessary. Document the optimal conditions thoroughly to ensure reproducibility in future experiments.

What are the best experimental designs for studying BHLH113 interactions with other proteins?

Studying protein-protein interactions involving BHLH113 requires careful experimental design. Based on approaches used to study other bHLH family members, several complementary methods can be employed:

• Co-immunoprecipitation (Co-IP): This technique can directly assess protein interactions in near-native conditions. Express BHLH113 with potential interaction partners (with different tags) in an appropriate system, then perform reciprocal Co-IPs using antibodies against each protein . Including appropriate controls (such as IgG control and non-interacting protein controls) is essential for interpreting results.

• Split-GFP or BiFC assays: These methods visualize protein interactions in living cells. For example, a tripartite split-GFP system can be used where BHLH113 and its potential interacting partner are fused to complementary GFP fragments. If the proteins interact, functional GFP is reconstituted, producing fluorescence .

• Yeast two-hybrid screening: This approach can identify novel interaction partners of BHLH113 from a library of proteins. Verification of these interactions should then be performed using the methods above.

• Proximity ligation assay (PLA): This technique can detect protein interactions with high sensitivity and provide spatial information about where in the cell these interactions occur.

• Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET): These techniques can provide real-time measurements of protein interactions in living cells and are particularly useful for studying dynamic interactions.

When designing these experiments, consider factors such as subcellular localization (nuclear vs. cytoplasmic), post-translational modifications, and the presence of cofactors that might influence interactions . Controls should include known interacting partners (positive control) and proteins unlikely to interact with BHLH113 (negative control).

What are common issues when using BHLH113 antibodies in western blotting and how can they be resolved?

Western blotting with BHLH113 antibodies can encounter several challenges. Here are common issues and their solutions:

Multiple bands or high background: This might indicate cross-reactivity or non-specific binding. Solutions include:

• Increasing blocking time or concentration (try 5% BSA instead of milk for phospho-specific antibodies)

• Using more stringent washing conditions (increase salt concentration in TBST)

• Diluting the primary antibody further

• Including a competitive peptide control to identify specific bands

• Optimizing transfer conditions for proteins in the size range of BHLH113

Weak or no signal: This could result from low protein expression, inefficient transfer, or antibody degradation. Try:

• Increasing protein loading (50-100 μg of total protein)

• Optimizing extraction methods for nuclear proteins like BHLH113

• Enhancing detection with signal amplification systems

• Using fresh antibody aliquots and avoiding repeated freeze-thaw cycles

• Confirming transfer efficiency with reversible staining methods

Inconsistent results between experiments: This often relates to variability in technique or reagents. Standardize:

• Sample preparation methods

• Protein quantification

• Gel percentage and running conditions

• Transfer parameters

• Detailed documentation of protocols

• Use of appropriate housekeeping controls

Incorrect molecular weight bands: BHLH113, like other transcription factors, may undergo post-translational modifications or exist in different isoforms. Verify:

• Expected molecular weight based on amino acid sequence plus modifications

• Use of appropriate molecular weight markers

• Comparison with recombinant protein controls

• Running gradient gels for better resolution of proteins in the relevant size range

Remember that buffer composition, blocking agents, and incubation times may need to be optimized specifically for BHLH113 antibodies, as different antibodies have unique requirements for optimal performance.

How can subcellular localization of BHLH113 be accurately determined using immunofluorescence?

Accurate determination of BHLH113 subcellular localization requires careful experimental design and appropriate controls. Based on studies of related bHLH proteins, consider these methodological approaches:

Sample preparation:

• Fixation method is critical - use 4% paraformaldehyde for preserving protein localization while maintaining antigen accessibility

• Permeabilization conditions should be optimized (0.1-0.5% Triton X-100 for nuclear proteins)

• Consider using multiple cell types or tissues, as localization may vary by context

Staining protocol:

• Use optimized antibody concentration (start with 2-5 μg/ml for mouse monoclonal antibodies)

• Include a nuclear counterstain (DAPI or Hoechst) to clearly delineate the nucleus

• Consider co-staining with markers for specific cellular compartments (e.g., nuclear lamins, ER markers)

• Use high-quality secondary antibodies with minimal cross-reactivity

Essential controls:

• Primary antibody omission control to assess secondary antibody specificity

• Cells with BHLH113 knockdown/knockout as negative controls

• Competitive peptide blocking to confirm antibody specificity

• Overexpression of tagged BHLH113 for comparison with endogenous staining patterns

Advanced techniques:

• Super-resolution microscopy can provide detailed subnuclear localization information

• Time-course experiments may reveal dynamic changes in localization in response to stimuli

• Live-cell imaging with fluorescently-tagged BHLH113 can complement fixed-cell immunofluorescence

• Correlation with biochemical fractionation results (nuclear/cytoplasmic extracts followed by western blotting)

When analyzing results, quantitative assessment of nuclear versus cytoplasmic distribution should be performed across multiple cells and experimental replicates. Similar to findings with BHLH11, BHLH113 may show context-dependent localization patterns, possibly influenced by interaction partners or cellular conditions .

What strategies can improve chromatin immunoprecipitation (ChIP) efficiency with BHLH113 antibodies?

Chromatin immunoprecipitation (ChIP) for transcription factors like BHLH113 presents specific challenges that can be addressed through careful optimization:

Crosslinking optimization:

• Test different formaldehyde concentrations (0.5-2%) and crosslinking times (5-20 minutes)

• For weak or transient interactions, consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Optimize crosslinking quenching with glycine (125-200 mM) to prevent over-fixation

Chromatin preparation:

• Sonication parameters must be empirically determined for each cell type to achieve fragments of 200-500 bp

• Verify fragment size by agarose gel electrophoresis before proceeding

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

• Save input samples (5-10% of chromatin) before immunoprecipitation

Immunoprecipitation conditions:

• Antibody amount should be titrated (2-10 μg per reaction) to determine optimal concentration

• Increase sensitivity by using antibody-bead conjugation before adding chromatin

• Consider longer incubation times (overnight at 4°C) to improve binding efficiency

• Use appropriate negative controls (IgG isotype control, non-target region)

Washing and elution:

• Implement stringent washing steps with increasing salt concentrations

• Use fresh buffers and adequate washing volumes

• Optimize elution conditions to efficiently release protein-DNA complexes

• Consider on-bead digestion protocols for specialized applications

PCR/qPCR optimization:

• Design primers for known or predicted BHLH113 binding sites based on consensus E-box sequences

• Include positive control primers targeting regions bound by related bHLH factors

• Include negative control primers for regions unlikely to be bound

• Normalize ChIP-qPCR data to input and IgG controls

For challenging transcription factors like BHLH113, consider using epitope-tagged versions (HA, FLAG) expressed at near-endogenous levels if antibody performance is suboptimal. This approach can take advantage of highly specific anti-tag antibodies while maintaining biological relevance .

How should researchers evaluate BHLH113 antibody specificity across different experimental systems?

Comprehensive evaluation of BHLH113 antibody specificity requires testing across multiple experimental systems and employing orthogonal validation approaches. Following the recommendations of the academic and pharmaceutical histopathology consortium , consider this systematic validation strategy:

Multiple application testing:

• Begin with western blot analysis in various cell types and tissues

• Compare results across different applications (IHC, IF, IP, ChIP)

• Evaluate consistency of detection patterns across applications

Genetic model validation:

• Test antibody reactivity in BHLH113 knockout/knockdown models

• Compare wild-type versus modified samples in multiple applications

• Quantify signal reduction proportional to expression decrease

• Consider inducible systems for controlled expression levels

Cross-species reactivity assessment:

• Analyze antibody performance across relevant model organisms

• Align epitope sequences across species to predict reactivity

• Validate predicted reactivity experimentally

• Document species-specific optimization requirements

Epitope-specific validation:

• Perform peptide competition assays using the immunizing peptide

• Test antibody against recombinant BHLH113 fragments

• Compare antibodies targeting different BHLH113 epitopes

• Evaluate potential cross-reactivity with related bHLH family members

Orthogonal technique comparison:

• Correlate antibody-based detection with mRNA expression data

• Compare results with mass spectrometry-based protein identification

• Validate with orthogonal detection methods (e.g., RNA-seq, proteomics)

• Document concordance and discrepancies between methods

Following this tiered approach to validation creates a comprehensive profile of antibody performance that should be documented and shared with the scientific community. Researchers should consider placing BHLH113 antibodies into appropriate tiers (1-3) based on validation evidence, with higher tiers indicating more thoroughly validated reagents suitable for critical applications .

What controls are essential when conducting immunoprecipitation experiments with BHLH113 antibodies?

Immunoprecipitation (IP) experiments with BHLH113 antibodies require rigorous controls to ensure reliable and interpretable results. Based on best practices for similar transcription factor studies, include the following essential controls:

Input control:

• Save 2-10% of the pre-IP lysate as input control

• Process in parallel with IP samples (except for the IP step)

• Use to normalize IP efficiency and verify protein presence in starting material

• Essential for quantitative comparisons between samples

Negative controls:

• Isotype-matched non-specific IgG control performed under identical conditions

• IP using the same antibody in cells lacking BHLH113 expression

• Pre-clearing beads-only control to assess non-specific binding to beads

• Pre-immune serum control when using polyclonal antibodies

Specificity controls:

• Competitive peptide blocking using the immunizing peptide

• Comparison of multiple antibodies targeting different BHLH113 epitopes

• IP using tagged BHLH113 with both anti-tag and anti-BHLH113 antibodies

• Reciprocal IP of known interaction partners (similar to approaches used for BHLH11 and BHLH IVc proteins)

Technical controls:

• Verify antibody orientation in antibody-bead complexes (direct vs. sandwich)

• Test different lysis buffers to optimize protein extraction and maintain interactions

• Include protease and phosphatase inhibitors to prevent degradation

• Control for antibody heavy and light chains in western blot detection

When studying interactions between BHLH113 and other proteins, use stringent washing conditions to remove non-specific interactions while maintaining true binding partners. Sequential immunoprecipitation (re-IP) can provide strong evidence for the existence of specific protein complexes containing BHLH113 .

For Co-IP experiments specifically, demonstrate interaction specificity by showing that the interaction is resistant to increased salt concentration (up to 300 mM NaCl) but can be disrupted under more stringent conditions. Additionally, verify that the interaction can be recapitulated with recombinant proteins in vitro when feasible.

How can researchers effectively distinguish between BHLH113 and other closely related bHLH family members?

Distinguishing BHLH113 from other closely related bHLH family members requires strategic approaches that leverage unique characteristics of each protein. Consider these specialized techniques:

Antibody-based discrimination:

• Select antibodies targeting non-conserved regions outside the bHLH domain

• Perform extensive cross-reactivity testing against related bHLH proteins

• Use epitope mapping to confirm antibody recognition sites

• Employ competitive binding assays with specific peptides representing unique regions

Expression pattern analysis:

• Compare tissue-specific or cell-type-specific expression profiles of bHLH family members

• Use qRT-PCR with highly specific primers to differentiate mRNA expression

• Correlate protein detection with known expression patterns

• Leverage single-cell techniques to resolve expression in heterogeneous samples

Functional differentiation:

• Analyze DNA binding site preferences through ChIP-seq or in vitro binding assays

• Compare interaction partners through Co-IP followed by mass spectrometry

• Assess transcriptional activation/repression activity in reporter assays

• Evaluate post-translational modifications specific to BHLH113

Genetic approaches:

• Use CRISPR/Cas9 to specifically tag endogenous BHLH113 with an epitope tag

• Generate knockout models for individual bHLH proteins

• Perform rescue experiments with specific bHLH members

• Utilize selective degradation approaches (e.g., auxin-inducible degron systems)

Biophysical techniques:

• Compare protein molecular weights by SDS-PAGE with high-resolution gels

• Utilize 2D gel electrophoresis to separate based on both isoelectric point and molecular weight

• Apply mass spectrometry to identify unique peptide sequences

• Use structural analysis (if available) to highlight distinguishing features

When working with antibodies recognizing multiple bHLH proteins, consider using complementary approaches, such as RNA interference to selectively deplete individual bHLH members and observe changes in antibody reactivity patterns. This approach can help determine the contribution of each family member to the observed signal, similar to studies performed with BHLH11 and related proteins .