BHLH109 Antibody

What is BHLH109 and why are antibodies against it important for plant research?

BHLH109 is a basic helix-loop-helix transcription factor involved in somatic embryogenesis (SE) in plants. Antibodies against BHLH109 are crucial tools for studying its expression patterns, protein interactions, and functional roles during embryogenic development.

Recent studies indicate that BHLH109 exhibits a differential and auxin-dependent pattern of expression during embryogenic culture, with reduced transcription in non-embryogenic callus tissue and low activity in tissue induced towards shoot regeneration via organogenesis . These characteristics make BHLH109 a significant target for researchers studying plant developmental pathways.

How are BHLH proteins structured and what functional domains should antibodies target?

BHLH proteins contain a basic domain that binds DNA and a helix-loop-helix domain that facilitates protein-protein interactions. When designing or selecting antibodies against BHLH109, researchers should consider:

• The C-terminal domain, which is often critical for protein-protein interactions (as seen with related family member MdbHLH104)

• Unique epitopes that distinguish BHLH109 from other BHLH family members

• Functional domains involved in transcriptional regulation

Different domains serve different functions, and antibodies targeting specific regions can yield different experimental outcomes. For co-immunoprecipitation experiments, antibodies targeting interaction domains may be more effective than those targeting DNA-binding regions.

What validation methods should be used specifically for BHLH109 antibodies?

Thorough validation of BHLH109 antibodies is essential using multiple complementary approaches:

Recent studies in antibody characterization emphasize that validation data are potentially cell or tissue type specific, requiring characterization to be performed for each specific use .

How can researchers distinguish between specific and non-specific binding when using BHLH109 antibodies?

To distinguish between specific and non-specific binding:

• Perform pre-adsorption tests with purified recombinant BHLH109 protein

• Include appropriate negative controls (knockout/knockdown tissue)

• Conduct competitive binding assays with known BHLH109 interacting proteins

• Use knockout cell lines which have shown to be superior to other types of controls for Western Blots and immunofluorescence imaging

• Include blocking controls with unrelated proteins of similar structure

When interpreting results, be aware that even carefully validated antibodies may show contextual specificity changes in different experimental conditions or tissue types .

What is the optimal protocol for immunoprecipitation of BHLH109 and its interaction partners?

For effective co-immunoprecipitation of BHLH109 and interaction partners:

• Extract proteins using buffer containing:

  • 20 mM Tris (pH 7.4)

  • 100 mM NaCl

  • 0.5% Nonidet P-40

  • 0.5 mM EDTA

  • 0.5 mM phenylmethylsulfonyl fluoride

  • 0.5% Protease Inhibitor Cocktail

• Pre-clean 1 mg of protein extract with Protein A agarose beads (4h, 4°C)

• Centrifuge and transfer supernatant to a fresh tube

• Incubate with anti-BHLH109 antibody overnight at 4°C

• Wash precipitates four times

• Add loading buffer and analyze by SDS-PAGE and western blotting

For detecting potential ubiquitination of BHLH109 (similar to MdbHLH104), include the proteasome inhibitor MG132 in your treatments prior to protein extraction to prevent degradation of ubiquitinated proteins .

How should immunocytochemical analysis be performed to visualize BHLH109 localization in plant tissues?

For effective immunocytochemical visualization of BHLH109:

• Begin with appropriate fixation:

  • Use 4% paraformaldehyde for structural preservation

  • Embed samples in appropriate medium and section

• Apply blocking buffer:

  • 2% fetal calf serum and 2% bovine serum albumin in PBS for 30 min at room temperature

• Apply primary BHLH109 antibody:

  • Dilute 1:20 in blocking buffer

  • Incubate overnight at 4°C

• Wash samples three times in blocking buffer

• Apply secondary antibody:

  • Use fluorescently labeled secondary antibodies (e.g., AlexaFluor 488 anti-rabbit IgG)

  • Dilute 1:100 in blocking buffer

  • Incubate for 1.5h at room temperature

• Wash and counterstain as appropriate

• Image using epifluorescence or confocal microscopy

Always include appropriate controls, including sections incubated without primary antibody and sections from plants with altered BHLH109 expression.

What are common causes of false positive or negative results with BHLH109 antibodies and how can they be addressed?

Common issues with BHLH109 antibodies and their solutions:

Keep in mind that approximately 50% of commercial antibodies fail to meet basic standards for characterization , making thorough validation essential.

How should researchers interpret contradictory results between antibody-based detection of BHLH109 and gene expression data?

When facing contradictions between protein detection and gene expression:

• Consider post-transcriptional regulation:

  • BHLH109 protein may be regulated by ubiquitination and proteasomal degradation (similar to MdbHLH104)

  • Protein stability may vary across developmental stages or conditions

• Evaluate antibody specificity:

  • Reconfirm antibody validation with genetic knockouts/knockdowns

  • Test whether post-translational modifications affect antibody recognition

• Examine experimental conditions:

  • Protein extraction methods might affect detection efficiency

  • Different fixation protocols can influence epitope accessibility

• Employ orthogonal methods:

  • Use tagged BHLH109 constructs to verify antibody detection

  • Apply absolute quantification methods for both protein and mRNA

Remember that protein abundance does not always correlate with mRNA levels due to differences in translation efficiency, protein stability, and post-translational regulation.

How can researchers investigate potential post-translational modifications of BHLH109 using antibody-based approaches?

To investigate post-translational modifications (PTMs) of BHLH109:

• For ubiquitination studies:

  • Perform immunoprecipitation with BHLH109 antibody

  • Immunoblot with anti-ubiquitin antibody

  • Include proteasome inhibitors (e.g., MG132) in extractions

  • Consider using cell-free degradation assays as performed with MdbHLH104

• For phosphorylation analysis:

  • Use phospho-specific antibodies if available

  • Combine with phosphatase treatments as controls

  • Consider Phos-tag SDS-PAGE for mobility shift detection

• For interaction with modification machinery:

  • Investigate interactions with E3 ligases (similar to BTB-TAZ proteins that interact with MdbHLH104)

  • Use co-immunoprecipitation followed by mass spectrometry

• For functional impact of modifications:

  • Compare wild-type and modification-resistant mutant variants

  • Assess protein stability and function in response to stimuli

These approaches can reveal how BHLH109 activity is regulated post-translationally during plant development and in response to environmental signals.

What are the most effective strategies for developing recombinant antibodies against BHLH109 with improved specificity?

For developing improved recombinant antibodies against BHLH109:

• Antigen design considerations:

  • Focus on unique regions that distinguish BHLH109 from other family members

  • Consider both linear epitopes and conformational determinants

  • Express multiple fragments of the protein to target different domains

• Selection technologies:

  • Use phage display with stringent negative selection against related BHLH proteins

  • Apply yeast display for higher throughput screening

  • Consider directed evolution approaches to enhance specificity

• Characterization requirements:

  • Employ knockout validation (gold standard)

  • Use multiple orthogonal approaches across different applications

  • Test cross-reactivity against closely related BHLH family members

• Recombinant antibody formats:

  • Single-chain variable fragments (scFvs) for better tissue penetration

  • Full-length IgG formats for applications requiring Fc-mediated functions

  • Multi-specific formats targeting multiple epitopes simultaneously

Recent advances have shown that recombinant antibodies consistently outperform both monoclonal and polyclonal antibodies across various assays .

How can BHLH109 antibodies be employed to study protein-DNA interactions in chromatin contexts?

To study BHLH109-DNA interactions in chromatin:

• Chromatin Immunoprecipitation (ChIP) approaches:

  • Optimize crosslinking conditions for plant tissues

  • Ensure antibody specifically recognizes BHLH109 in crosslinked chromatin

  • Consider ChIP-seq to identify genome-wide binding sites

  • Use appropriate controls (input DNA, IgG control, knockout tissue)

• For sequential ChIP (Re-ChIP) to study transcriptional complexes:

  • First immunoprecipitate with BHLH109 antibody

  • Re-immunoprecipitate with antibodies against suspected cofactors

  • This can reveal co-occupancy on specific genomic regions

• For monitoring binding dynamics:

  • Combine with time-course experiments during developmental processes

  • Correlate with expression analysis of target genes

• For validation of binding sites:

  • Use electrophoretic mobility shift assays (EMSA) with recombinant BHLH109

  • Employ reporter gene assays with putative target sequences

These approaches can establish direct links between BHLH109 binding and the regulation of target genes involved in somatic embryogenesis and other developmental processes.