BHLH133 Antibody

What is BHLH133 and how does it relate to other bHLH transcription factors?

BHLH133 belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors, which play crucial roles in various biological processes. The bHLH family is categorized into multiple subgroups, with members showing varying degrees of functional redundancy. For context, the bHLH subgroup IIId factors (including bHLH3, bHLH13, bHLH14, and bHLH17) function as transcription repressors that negatively regulate jasmonate (JA) responses in plants . These factors show high amino acid similarity and functional redundancy, as demonstrated by progressively enhanced phenotypes in multiple mutants .

BHLH133, while not specifically characterized in the provided search results, would be expected to share structural features with other bHLH proteins, including the characteristic basic DNA-binding domain and the helix-loop-helix domain involved in protein-protein interactions. Research into related bHLH factors has shown they are expressed in various plant tissues and can be localized in the nucleus or in both the nucleus and cytoplasm, depending on the specific factor .

What are the typical applications of BHLH133 antibodies in research?

BHLH133 antibodies can be utilized in numerous research applications, including:

• Protein detection and quantification: Western blotting to detect BHLH133 expression levels in different tissues or under various experimental conditions.

• Subcellular localization studies: Immunofluorescence microscopy to determine where BHLH133 is located within cells, similar to the nuclear and cytoplasmic localizations observed for other bHLH factors .

• Protein-protein interaction studies: Co-immunoprecipitation to identify binding partners of BHLH133.

• Chromatin immunoprecipitation (ChIP): To identify DNA-binding sites and target genes of BHLH133.

• Tissue expression profiling: Immunohistochemistry to map expression patterns across different tissues, similar to the approaches used for other bHLH factors using GUS reporter systems .

How is antibody specificity validated for transcription factors like BHLH133?

Validating antibody specificity for transcription factors requires a multi-faceted approach:

• Western blot analysis: Using wildtype samples alongside knockout/knockdown controls to confirm the antibody detects a band of the expected size that is absent or reduced in the negative control.

• Immunoprecipitation followed by mass spectrometry: To confirm the antibody pulls down the intended protein.

• Recombinant protein testing: Testing against purified recombinant BHLH133 protein versus other bHLH family members to assess cross-reactivity.

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish specific binding.

• Genetic validation: Testing antibody reactivity in tissues from knockout/knockdown models, similar to the genetic approach used to study bHLH factor functions through analysis of single, double, triple, and quadruple mutants .

The validation approach should be systematic and thorough, particularly given the high sequence similarity among bHLH family members that could lead to cross-reactivity issues.

What molecular features should be considered when designing experiments to study BHLH133-DNA interactions?

When designing experiments to study BHLH133-DNA interactions, researchers should consider:

• DNA-binding domain characteristics: The basic region of bHLH proteins recognizes specific DNA sequences. Understanding the precise binding motif for BHLH133 is crucial for experimental design.

• Dimerization potential: bHLH proteins often form homo- or heterodimers through their HLH domains. Experiments should account for possible interactions with other bHLH family members that might affect DNA binding.

• Post-translational modifications: These can affect DNA-binding affinity. Experimental conditions should preserve relevant modifications.

• Chromatin context: In vivo binding may depend on chromatin accessibility and cooperation with other factors.

• Nuclear localization: As observed with other bHLH factors, subcellular localization can vary (e.g., bHLH3 and bHLH17 are nucleus-localized, while bHLH13 and bHLH14 are found in both nucleus and cytoplasm) . This differential localization affects experimental design for studying DNA interactions.

• Experimental approach selection: Choose between in vitro (EMSA, DNA footprinting) and in vivo (ChIP-seq) methods based on your specific research questions.

How can biolayer interferometry be optimized for characterizing BHLH133 antibody binding kinetics?

Biolayer interferometry (BLI) can be optimized for BHLH133 antibody characterization based on protocols used for other antibodies:

• Immobilization strategy:

  • Capture the BHLH133 antibody on anti-human IgG Fc biosensors

  • Alternatively, immobilize purified BHLH133 protein and probe with antibody

  • Test both orientations to determine optimal signal-to-noise ratio

• Buffer optimization:

  • Use protein-free blocking buffer with PBS and 0.5% (v/v) Tween-20 to minimize non-specific binding

  • Maintain consistent pH and ionic strength across experiments

• Concentration ranges:

  • Test a series of at least seven concentrations of antigen or antibody

  • For initial experiments, start with concentrations ranging from 5-100 nM

• Association/dissociation times:

  • Allow sufficient time for association (300-600 seconds) and dissociation (700-1200 seconds) to obtain reliable kinetic parameters

• Data analysis:

  • Apply a 1:1 binding model for initial analysis

  • Consider more complex binding models if simple models provide poor fits

  • Include reference subtraction and alignment in analysis software

• Controls:

  • Include biosensors with non-specific antibodies to test for potential non-specific interactions

  • Use purified recombinant protein versus cell lysates containing BHLH133

What strategies can address epitope masking when studying BHLH133 in protein complexes?

Epitope masking occurs when the antibody binding site becomes inaccessible due to protein-protein interactions or conformational changes. To address this challenge:

• Multiple antibody approach: Develop antibodies targeting different epitopes on BHLH133, as protein complexes may mask some regions while leaving others accessible.

• Mild denaturation protocols: Apply gentle denaturation conditions that disrupt protein complexes while preserving epitope structure.

• Cross-linking strategies: For interaction studies, use membrane-permeable cross-linkers to capture transient interactions before cell lysis.

• Epitope mapping: Determine precisely which amino acid sequences are recognized by the antibody to predict potential masking scenarios.

• Alternative detection methods: Complement antibody-based detection with tagged BHLH133 constructs in cases where epitope masking is unavoidable.

• Native vs. denaturing conditions: Compare antibody performance under both conditions to identify potential masking effects, especially important for bHLH proteins that form dimers through their HLH domains.

What experimental design principles should guide BHLH133 antibody validation studies?

A robust experimental design for BHLH133 antibody validation should follow these principles:

• Clear hypothesis formulation: Explicitly state the expected antibody performance characteristics (specificity, sensitivity, reproducibility).

• Variable identification and control:

  Variable Type	Examples	Control Method
  Independent	Antibody concentration, incubation time	Systematic variation
  Dependent	Signal intensity, background	Quantitative measurement
  Extraneous	Temperature fluctuations, sample handling	Standardized protocols
  Confounding	Cross-reactivity with other bHLH proteins	Include related protein controls

• Between-subjects or within-subjects design: For comparing different antibody lots or clones, a within-subjects design using the same samples across tests minimizes sample variation .

• Sample size calculation: Determine appropriate technical and biological replicates based on expected variation.

• Controls inclusion:

  • Positive controls: Samples with confirmed BHLH133 expression

  • Negative controls: Knockout/knockdown samples, pre-immune serum

  • Specificity controls: Related bHLH proteins to assess cross-reactivity

• Randomization: Randomize the order of sample processing to minimize systematic errors.

• Blinding: When feasible, blind the researcher to sample identity during analysis to prevent confirmation bias .

How can structural insights inform the development of high-specificity antibodies against BHLH133?

Structural approaches can significantly enhance antibody development against transcription factors like BHLH133:

• Epitope selection based on structural uniqueness:

  • Target regions that diverge from other bHLH family members

  • Focus on surface-exposed loops rather than conserved DNA-binding domains

• Somatic hypermutation considerations:

  • Somatic hypermutations shape the complementarity-determining regions (CDRs) of antibodies, critically affecting binding specificity

  • For hybridoma development, screen multiple clones to identify those with optimal mutation patterns

• Structural modeling for epitope prediction:

  • Use homology modeling based on related bHLH structures to predict BHLH133 structure

  • Identify unique surface features for epitope targeting

• Complementarity optimization:

  • Design antibodies with complementary charge distributions to the target epitope

  • Small modifications can significantly impact binding, as seen with the D93N mutation in the 3I14 antibody that improved binding affinity through enhanced charge complementarity and hydrogen bonding

• Cross-reactivity minimization:

  • Analyze potential cross-reactive epitopes through structural alignment of bHLH family members

  • Pre-absorb antibodies against related proteins

What are the recommended troubleshooting approaches for non-specific binding in BHLH133 immunoprecipitation experiments?

When facing non-specific binding issues in BHLH133 immunoprecipitation experiments, consider these methodical troubleshooting approaches:

• Buffer optimization:

  • Adjust salt concentration incrementally (150-500 mM)

  • Test different detergent types and concentrations

  • Modify pH conditions to optimize antibody-antigen interaction while minimizing non-specific binding

• Blocking optimization:

  • Compare different blocking agents (BSA, non-fat milk, commercial blockers)

  • Pre-clear lysates with beads alone before immunoprecipitation

  • Use appropriate concentration of non-specific IgG from the same species

• Cross-linking optimization:

  • If using cross-linking, titrate cross-linker concentration

  • Optimize cross-linking time to capture specific interactions without excessive non-specific aggregation

• Systematic troubleshooting protocol:

  Issue	Potential Cause	Troubleshooting Approach
  High background	Insufficient washing	Increase wash stringency gradually
  Multiple bands	Cross-reactivity	Perform peptide competition assay
  No signal	Epitope denaturation	Try native conditions
  Inconsistent results	Variable BHLH133 expression	Normalize lysate input

• Advanced approaches:

  • Tandem purification using two different epitopes

  • Comparison of different antibody clones

  • Mass spectrometry validation of immunoprecipitated proteins

How can BHLH133 antibodies be effectively utilized in chromatin immunoprecipitation experiments?

To effectively utilize BHLH133 antibodies in ChIP experiments:

• Cross-linking optimization:

  • Titrate formaldehyde concentration (typically 0.75-1.5%)

  • Optimize cross-linking time (8-15 minutes) to efficiently capture DNA-protein interactions without over-fixation

• Chromatin fragmentation:

  • Sonicate to achieve DNA fragments of 200-500 bp

  • Verify fragmentation efficiency by running samples on agarose gels

• Antibody validation for ChIP:

  • Test antibody in immunoprecipitation of native protein

  • Confirm ability to recognize fixed protein

  • Verify enrichment of known or predicted target sequences

• Controls:

  • Include IgG negative control

  • Use input chromatin as reference

  • Include positive control (known target gene) and negative control regions

  • When available, use BHLH133 knockout/knockdown samples as specificity controls

• Optimization for low abundance factors:

  • Increase starting material

  • Reduce background with stringent washing

  • Consider ChIP-exo or ChIP-nexus for higher resolution

• Data analysis considerations:

  • Normalize to input

  • Apply appropriate statistical tests

  • Validate findings with orthogonal methods (e.g., reporter assays)

What considerations are important when using BHLH133 antibodies for detecting protein-protein interactions?

When using BHLH133 antibodies to study protein-protein interactions:

• Interaction preservation:

  • Choose lysis conditions that maintain protein interactions (mild non-ionic detergents, physiological salt)

  • Consider crosslinking for transient interactions

  • Maintain appropriate buffer pH and ionic strength

• Reciprocal co-immunoprecipitation:

  • Confirm interactions by immunoprecipitating with antibodies against both BHLH133 and its suspected partner

  • This approach helps validate true interactions and rule out non-specific binding

• Epitope accessibility assessment:

  • Test whether the antibody can access its epitope when BHLH133 is in complex with partners

  • Similar to how other bHLH factors form complexes that might affect epitope accessibility

• Controls for specificity:

  • Include non-specific IgG controls

  • Use cells not expressing BHLH133 or potential partners

  • Include competition with blocking peptides

• Interaction validation techniques:

  • Support co-IP findings with orthogonal methods:

    • Proximity ligation assay

    • FRET/BRET

    • Yeast two-hybrid

    • GST pull-down with recombinant proteins

• Interpretation caveats:

  • Consider whether interactions are direct or mediated by other proteins

  • Assess potential effects of antibody binding on interaction interfaces

  • Evaluate physiological relevance of detected interactions

How do post-translational modifications impact BHLH133 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of BHLH133:

• Common PTMs affecting transcription factors:

  • Phosphorylation of serine/threonine/tyrosine residues

  • Acetylation of lysine residues

  • Methylation, SUMOylation, and ubiquitination

  These modifications can alter protein conformation and epitope accessibility.

• Epitope-specific considerations:

  • Antibodies raised against unmodified peptides may fail to recognize modified forms

  • Conversely, modification-specific antibodies will only detect the modified subpopulation

• Modification-sensitive experimental strategies:

  • Use phosphatase treatment to remove phosphorylations

  • Compare results with modification-specific and pan-BHLH133 antibodies

  • Consider using multiple antibodies targeting different epitopes

• Functional implications:

  • PTMs may regulate BHLH133 activity, DNA binding, protein-protein interactions, and subcellular localization

  • Similar to how other bHLH factors show differential subcellular localization patterns

• Technical approaches for PTM analysis:

  • 2D gel electrophoresis to separate modified forms

  • Phos-tag gels for phosphorylation detection

  • Mass spectrometry to identify and map modifications

What are the critical factors in designing experiments to study BHLH133 protein half-life and degradation?

When designing experiments to study BHLH133 protein half-life and degradation:

• Protein synthesis inhibition approach:

  • Use cycloheximide to block new protein synthesis

  • Collect samples at multiple timepoints (0, 1, 2, 4, 8, 16, 24 hours)

  • Quantify BHLH133 levels by Western blot

• Degradation pathway inhibitors:

  • Test proteasome inhibitors (MG132, bortezomib)

  • Test lysosome inhibitors (chloroquine, bafilomycin A1)

  • Compare effects to determine predominant degradation pathway

• Pulse-chase experiments:

  • Label proteins with 35S-methionine (radioactive) or click chemistry (non-radioactive)

  • Chase with non-labeled amino acids

  • Immunoprecipitate BHLH133 at different timepoints

• Ubiquitination detection:

  • Co-immunoprecipitate BHLH133 and blot for ubiquitin

  • Use TUBEs (tandem ubiquitin binding entities) to enrich ubiquitinated proteins

• Experimental design considerations:

  Variable	Consideration	Control Method
  Cell density	Affects growth rate and metabolism	Standardize seeding density
  Transfection efficiency	Varies between experiments	Include transfection control
  Protein expression level	Can affect degradation kinetics	Compare endogenous and overexpressed
  Treatment toxicity	May cause non-specific effects	Monitor cell viability

• Data analysis:

  • Plot protein levels against time on semi-log scale

  • Calculate half-life using first-order decay kinetics

  • Compare treatments using appropriate statistical tests