BHLH118 Antibody

What is BHLH118 and why is it significant in plant research?

BHLH118 (Basic Helix-Loop-Helix protein 118), also known as EN5, is a transcription factor belonging to the bHLH family found in Arabidopsis thaliana. The significance of BHLH118 stems from its role as a transcriptional regulator that binds specific DNA sequences through its bHLH domain. bHLH transcription factors are known to recognize specific DNA motifs including E-box elements (5'-CANNTG-3') .

The bHLH family plays crucial regulatory roles in various plant developmental processes and stress responses. While specific BHLH118 functions are still being elucidated, research on related bHLH proteins suggests involvement in numerous biological processes including flavonoid biosynthesis, iron homeostasis, and developmental regulation . The antibody against this protein enables researchers to study its expression patterns, subcellular localization, and potential protein-protein interactions.

What are the technical specifications of commercially available BHLH118 antibodies?

Currently available BHLH118 antibodies include:

These antibodies are primarily designed for research applications and should not be used for diagnostic or therapeutic procedures .

How should I optimize Western blot protocols for BHLH118 detection in plant samples?

For optimal Western blot detection of BHLH118 in plant samples:

• Sample preparation:

  • Grind plant tissue to a fine powder in liquid nitrogen

  • Extract proteins using RIPA buffer (50mM Tris, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM PMSF, 1× protease inhibitor cocktail, pH 8.0)

  • Include phosphatase inhibitors if investigating potential phosphorylation states

• Nuclear protein enrichment: Since BHLH118 is primarily nuclear-localized, enrich nuclear proteins to increase detection sensitivity:

  • Follow nuclear/cytoplasmic fractionation protocols similar to those used for other bHLH proteins

  • Use this approach for samples with low BHLH118 expression

• Gel electrophoresis and transfer:

  • Use 12% SDS-PAGE gels for optimal resolution

  • Transfer to nitrocellulose membranes at 100V for 60-90 minutes

• Blocking and antibody incubation:

  • Block with TBST (10mM Tris-Cl, 150mM NaCl, 0.05% Tween-20, pH 8.0) containing 5% nonfat milk

  • Incubate with BHLH118 antibody at 1:1000-1:2000 dilution overnight at 4°C

  • Wash with TBST (3 times for 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody at room temperature for 1.5 hours

  • Visualize with enhanced chemiluminescence (ECL) substrate

• Controls:

  • Include positive control (Arabidopsis thaliana wild-type extract)

  • Include negative control (if available, extract from bhlh118 knockout line)

  • Use loading control (actin or tubulin) to normalize protein loading

What methods can I use to study protein-protein interactions involving BHLH118?

Based on methods successfully used for other bHLH family proteins, the following approaches are recommended:

• Yeast Two-Hybrid (Y2H):

  • Clone BHLH118 into a bait vector (e.g., pGBKT7 with GAL4 DNA binding domain)

  • Screen against a prey library or specific candidate interactors

  • Verify positive interactions with additional assays

• Split-GFP/BiFC assays:

  • For tripartite split-GFP: Fuse BHLH118 to GFP11 fragment and potential interactors to GFP10

  • Co-express with GFP1-9 fragment in plant cells (e.g., Nicotiana benthamiana leaves)

  • Visualize reconstituted GFP fluorescence by confocal microscopy

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of BHLH118 (e.g., HA-BHLH118) and potential interactor proteins (e.g., MYC-tagged)

  • Extract proteins from transfected plant tissues in IP buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM MgCl₂, 20% glycerol, 0.2% NP-40, protease/phosphatase inhibitors)

  • Immunoprecipitate using antibody against one tag

  • Detect co-precipitated proteins by Western blot using antibody against the other tag

• Proximity Labeling approaches:

  • Fuse BHLH118 to BioID or TurboID

  • Identify proximal proteins through biotin labeling and streptavidin pull-down

  • Analyze interactome by mass spectrometry

These methods complement each other and should be used in combination for robust verification of interaction partners.

How can I determine if my BHLH118 antibody is specific for its target protein?

Validating antibody specificity is crucial for reliable research outcomes. For BHLH118 antibody:

• Western blot validation:

  • Compare wild-type Arabidopsis extract with bhlh118 knockout/knockdown line

  • Expected result: Detection of a band of correct molecular weight in wild-type that is absent or reduced in knockout/knockdown

  • Consider performing peptide competition assay to confirm specificity

• Immunoprecipitation-mass spectrometry:

  • Perform IP with the BHLH118 antibody

  • Analyze precipitated proteins by mass spectrometry

  • BHLH118 should be among the most abundant proteins identified

• Recombinant protein controls:

  • Test antibody against purified recombinant BHLH118 protein

  • Test against closely related bHLH family members to assess cross-reactivity

• Cross-reference with orthogonal methods:

  • Compare protein expression with mRNA expression data

  • Use epitope-tagged BHLH118 expressed in plants and detect with both anti-tag and anti-BHLH118 antibodies

As noted in comprehensive antibody validation studies, using knockout cell lines as negative controls is considered superior to other types of controls, particularly for Western blot and immunofluorescence applications .

How can I use BHLH118 antibody to investigate DNA-binding properties and transcriptional regulation?

To study the DNA-binding and transcriptional regulatory functions of BHLH118:

• Chromatin Immunoprecipitation (ChIP):

  • Crosslink protein-DNA complexes in plant tissues

  • Sonicate to fragment chromatin

  • Immunoprecipitate BHLH118-bound DNA fragments using the BHLH118 antibody

  • Analyze pulled-down DNA by qPCR for candidate targets or ChIP-seq for genome-wide binding

  • Look for enrichment of E-box motifs (5'-CANNTG-3') or other known bHLH binding sites

• Electrophoretic Mobility Shift Assay (EMSA):

  • Design oligonucleotide probes containing predicted BHLH118 binding sites

  • Incubate with nuclear extracts containing BHLH118

  • Add BHLH118 antibody for supershift assay to confirm binding specificity

  • Compare binding patterns with those of other bHLH proteins that recognize different core sequences within E-box motifs

• Transactivation assays:

  • Clone promoters of putative target genes upstream of a reporter gene (e.g., luciferase)

  • Co-express with BHLH118 in protoplasts or plant cells

  • Measure reporter activity to assess transcriptional activation/repression

  • Use BHLH118 antibody in parallel experiments to confirm expression levels

• Identification of binding motif preferences:

  • Based on research with other bHLH proteins, determine if BHLH118 belongs to one of the three main clusters of bHLH proteins that recognize specific DNA half-sites: CAC, CAT, or CAG

  • Use high-throughput approaches like protein binding microarrays or HT-SELEX if available

How does BHLH118 compare structurally and functionally to other plant bHLH transcription factors?

BHLH118 belongs to the large family of bHLH transcription factors in Arabidopsis. When comparing it to other family members:

• Structural analysis:

  • The bHLH domain consists of approximately 60 amino acids with two functionally distinct regions: the basic region for DNA binding and the HLH region for dimerization

  • Phylogenetic analysis would place BHLH118 within one of the established subfamilies (likely among 18 subfamilies identified in related species)

  • Analysis of conserved motifs outside the bHLH domain can provide insights into functional specialization

• DNA binding specificity:

  • bHLH proteins can be categorized into three main clusters based on their DNA binding preferences:

    • Cluster 1: Recognizes CAC half-sites

    • Cluster 2: Recognizes CAT half-sites

    • Cluster 3: Recognizes CAG half-sites

  • Determining which cluster BHLH118 belongs to would provide insights into its potential target genes

• Dimerization properties:

  • Like other bHLH proteins, BHLH118 likely functions through homo- or hetero-dimerization

  • Investigating potential dimerization partners using methods outlined in section 2.2 can reveal functional networks

  • Some bHLH proteins act as antagonists to other family members, similar to the documented interaction between bHLH11 and bHLH IVc proteins

• Subcellular localization:

  • While primarily nuclear-localized, some bHLH proteins shuttle between cytoplasm and nucleus

  • Investigating whether BHLH118 shows dynamic localization under different conditions could reveal regulatory mechanisms

What methodological approaches can resolve contradictory experimental results with BHLH118 antibody?

When faced with contradictory results using BHLH118 antibody:

• Antibody batch variation:

  • Different lots of polyclonal antibodies may show variation in specificity and sensitivity

  • Solution: Request certificate of analysis for each lot and perform validation tests with each new batch

  • Consider using recombinant antibodies if available, as they typically show better reproducibility than polyclonal or even monoclonal antibodies

• Sample preparation inconsistencies:

  • BHLH118 detection may be sensitive to extraction methods or buffer compositions

  • Solution: Systematically compare nuclear extraction protocols to optimize recovery

  • Test multiple fixation methods for immunolocalization studies

• Protein expression dynamics:

  • BHLH118 expression may vary with developmental stage, tissue type, or environmental conditions

  • Solution: Perform time-course and tissue-specific analysis

  • Include positive controls from tissues/conditions known to express BHLH118

• Post-translational modifications:

  • Modifications may affect antibody recognition

  • Solution: Use phosphatase treatment or other enzymatic treatments to remove modifications

  • Consider using multiple antibodies recognizing different epitopes

• Technical validation:

  • Perform antibody characterization using strategies outlined in section 3.1

  • Include appropriate controls in each experiment

  • Use orthogonal methods (e.g., mass spectrometry, mRNA analysis) to confirm protein identity

Studies have shown that approximately 50-75% of proteins are covered by at least one high-performing commercial antibody, but performance varies by application. When selecting antibodies for specific applications, recombinant antibodies have been shown to outperform both monoclonal and polyclonal antibodies across multiple assay types .

How can I design experiments to elucidate the role of BHLH118 in plant stress responses and development?

To investigate BHLH118 function in plant biology:

• Expression analysis under various stress conditions:

  • Expose plants to different stresses (drought, salt, temperature, nutrient deficiency)

  • Analyze BHLH118 protein levels using the antibody in Western blot

  • Compare with transcriptional changes using qRT-PCR

  • Determine if stress affects BHLH118 subcellular localization using immunolocalization

• Genetic approaches:

  • Generate and characterize bhlh118 mutants and BHLH118 overexpression lines

  • Perform phenotypic analysis under normal and stress conditions

  • Use the antibody to confirm protein absence in knockout lines and overexpression in transgenic lines

  • Create lines expressing tagged versions of BHLH118 for ChIP-seq and interactome studies

• Metabolomic analysis:

  • Based on studies with other bHLH proteins, analyze changes in flavonoid profiles in bhlh118 mutants compared to wild-type

  • Use UPLC-ESI-MS/MS to identify differentially accumulated metabolites

  • Example dataset from a related bHLH protein study:

  Metabolite Class	Number of Compounds	Number Significantly Altered
  Anthocyanins	4	*
  Chalcones	2	*
  Dihydroflavonoids	5	*
  Flavonols	41	*
  Flavonoids	29	*
  Other classes	17	*
  Total identified	98	18

  *Specific numbers would depend on experimental results

• Transcriptome analysis:

  • Perform RNA-seq on bhlh118 mutants and overexpression lines

  • Identify differentially expressed genes

  • Combine with ChIP-seq data to distinguish direct from indirect targets

  • Look for enrichment of specific DNA motifs in promoters of regulated genes

• Protein-protein interaction network:

  • Identify BHLH118 interacting proteins using methods described in section 2.2

  • Confirm interactions using BHLH118 antibody in Co-IP experiments

  • Test whether interactions are modulated by environmental conditions

By integrating these approaches, researchers can build a comprehensive understanding of BHLH118 function in plant biology.

How might new antibody technologies improve BHLH118 research?

Emerging antibody technologies that could advance BHLH118 research include:

• Development of recombinant antibodies:

  • Recombinant antibodies offer superior reproducibility compared to polyclonal antibodies

  • Research shows recombinant antibodies outperform both monoclonal and polyclonal antibodies in multiple assays

  • Consider developing recombinant anti-BHLH118 antibodies using phage display or other modern antibody engineering approaches

• Nanobodies and single-domain antibodies:

  • Smaller antibody formats may offer improved access to nuclear proteins like BHLH118

  • Could enable live-cell imaging of BHLH118 dynamics when fused to fluorescent proteins

  • May provide better performance in ChIP applications

• Proximity labeling antibody conjugates:

  • Antibodies conjugated to enzymes like TurboID or APEX2

  • Would allow spatial proteomics to identify proteins in proximity to BHLH118 in situ

  • Could help map BHLH118 protein interaction networks with spatial resolution

• Enhanced brain delivery approaches:

  • While primarily relevant for mammalian research, innovations in antibody delivery systems may inspire new plant tissue penetration methods

  • Technologies like polymer conjugation could potentially enhance antibody delivery into dense plant tissues

These advanced approaches could significantly expand our ability to study BHLH118 biology in complex tissues and under dynamic conditions.

What are the most promising approaches for using BHLH118 antibody to understand transcription factor networks in plants?

To leverage BHLH118 antibody for understanding plant transcription factor networks:

• Sequential ChIP (Re-ChIP):

  • Perform ChIP with BHLH118 antibody followed by a second ChIP with antibodies against suspected co-factors

  • Identifies genomic regions bound by both BHLH118 and partner proteins

  • Reveals cooperative transcriptional regulation mechanisms

• Single-cell approaches:

  • Adapt antibody-based techniques for single-cell protein analysis

  • Could reveal cell-type specific BHLH118 expression patterns

  • Compare with single-cell transcriptomics data to identify cell-specific regulatory networks

• Multi-omics integration:

  • Combine antibody-based protein measurements with transcriptomics, metabolomics, and phenomics

  • Use systems biology approaches to model BHLH118 influence on plant physiology

  • Identify feedback loops and network motifs involving BHLH118

• CRISPR-based approaches combined with antibody validation:

  • Use CRISPR to tag endogenous BHLH118 for functional studies

  • Validate CRISPR modifications using BHLH118 antibody

  • Create precise mutations in DNA-binding or protein interaction domains to dissect BHLH118 function

• Cross-species conservation studies:

  • Use BHLH118 antibody to study orthologous proteins in different plant species

  • Test cross-reactivity with related proteins in crop species

  • Investigate conservation of regulatory networks across plant evolution

By combining these approaches, researchers can build a comprehensive understanding of how BHLH118 integrates into the broader regulatory landscape controlling plant development and stress responses.