BHLH80 Antibody

What is BHLH80 and what is its role in plant biology?

BHLH80 is a transcription factor belonging to the basic helix-loop-helix (bHLH) family. In Arabidopsis thaliana, it is also known by synonyms including EN71, AtbHLH80, and bHLH transcription factor bHLH080. Like other bHLH transcription factors, it recognizes and binds to E-box motifs (CANNTG) in DNA to regulate gene expression .

Research on EbbHLH80 from Erigeron breviscapus has shown that this transcription factor plays crucial roles in regulating flavonoid biosynthesis pathways. When overexpressed in tobacco, EbbHLH80 significantly increases flavonoid accumulation, with transgenic lines showing 1.41 to 1.49-fold higher flavonoid content compared to wild-type plants . Beyond flavonoid biosynthesis, EbbHLH80 appears to be involved in stress response mechanisms and hormone signal transduction pathways, including ABA and ethylene signaling .

What are the key characteristics of commercially available BHLH80 antibodies?

BHLH80 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana BHLH80 protein . These antibodies are affinity-purified and provided in liquid form. Key characteristics include:

These antibodies are designed for research use only and are not intended for diagnostic or therapeutic procedures .

How should researchers design experiments to study BHLH80's role in flavonoid biosynthesis?

Based on studies with EbbHLH80, researchers should consider the following experimental design:

• Gene expression analysis:

  • Quantify expression levels of BHLH80 across different plant tissues to establish baseline expression patterns

  • Studies with EbbHLH80 showed highest expression in leaves, correlating with high levels of flavonoids like scutellarin

• Functional validation through transgenic approaches:

  • Generate overexpression lines using a strong promoter (e.g., 35S)

  • Create knockout/knockdown lines using CRISPR-Cas9 or RNAi

  • Quantify total flavonoid content using spectrophotometric methods

  • Perform targeted metabolomics to identify specific flavonoid compounds affected

• Transcriptome analysis:

  • Compare gene expression profiles between wild-type and transgenic plants

  • Focus on key flavonoid biosynthesis genes: PAL, C4H, 4CL, CHS, CHI, FLS2, F3H, DFR, and ANS

  • Research with EbbHLH80 demonstrated upregulation of all these genes in overexpression lines

• Integration with other transcription factors:

  • Investigate interactions with MYB transcription factors and WD40 proteins

  • Previous research suggests BHLH factors often function in MYB-bHLH-WD40 complexes to regulate flavonoid biosynthesis

  • Examine potential crosstalk with ERF transcription factors, as 49 ERFs were found upregulated in EbbHLH80 overexpression lines

What is the optimal protocol for using BHLH80 antibody in Western blotting experiments?

For optimal Western blotting results with BHLH80 antibody:

• Sample preparation:

  • Harvest plant tissue and flash-freeze in liquid nitrogen

  • Grind tissue to fine powder and extract proteins using a buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1 mM EDTA

    • 1% Triton X-100

    • Protease inhibitor cocktail

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Quantify protein using Bradford or BCA assay

• SDS-PAGE separation:

  • Load 20-50 μg of protein per lane on 10-12% SDS-PAGE gel

  • Include positive control (e.g., recombinant BHLH80 or extract from overexpression line)

  • Include negative control (e.g., extract from knockout line if available)

• Transfer and blocking:

  • Transfer proteins to PVDF membrane (recommended over nitrocellulose for nuclear proteins)

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody incubation and detection:

  • Dilute BHLH80 antibody 1:1000 in blocking buffer

  • Incubate membrane overnight at 4°C with gentle agitation

  • Wash 3× with TBST, 10 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

  • Wash 3× with TBST, 10 minutes each

  • Develop using ECL substrate and detect signal

How can ChIP experiments be optimized using BHLH80 antibody to identify direct target genes?

For effective ChIP experiments with BHLH80 antibody:

• Chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes under vacuum

  • Quench with 0.125 M glycine for 5 minutes

  • Extract nuclei using a nuclear isolation buffer

  • Sonicate chromatin to generate fragments of 200-500 bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads

  • For each IP reaction, use 5 μg of BHLH80 antibody per 100 μg of chromatin

  • Include IgG control and input sample

  • Incubate overnight at 4°C with rotation

  • Add Protein A/G beads and incubate for 3 hours

  • Perform sequential washes with increasing stringency

• DNA recovery and analysis:

  • Reverse crosslinks at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using column purification

  • Perform qPCR analysis on known or predicted target genes

• Expected targets based on EbbHLH80 research:

  • Promoters of flavonoid biosynthesis genes (PAL, CHS, CHI, F3H, DFR, ANS)

  • Genes involved in hormone signaling pathways

  • Look for enrichment at E-box motifs (CANNTG)

How can researchers differentiate between BHLH80 and other closely related bHLH transcription factors?

Distinguishing BHLH80 from related bHLH factors requires multiple complementary approaches:

• Structural analysis:

  • Perform detailed sequence alignment focusing on the basic domain and DNA-binding residues

  • bHLH factors contain specific residues at positions 1, 2, 5, 6, 8, 9, 12 and 13 of the basic domain that make base-specific contacts with DNA

  • Analyze differences in these key residues to predict binding specificity

• DNA binding specificity:

  • While all bHLH factors recognize the core E-box motif (CANNTG), they show preferences for central nucleotides and flanking sequences

  • Perform in vitro binding assays (EMSA, protein binding microarrays) to determine sequence preferences

  • Compare ChIP-seq profiles to identify unique binding signatures

• Protein interaction partners:

  • Investigate specific protein-protein interactions through co-immunoprecipitation

  • bHLH factors form homo- and heterodimers with different partners

  • EbbHLH80 studies suggest interactions with ERF transcription factors that distinguish its regulatory network

• Functional validation:

  • Create transgenic lines with individual bHLH factors

  • Compare metabolic profiles, particularly flavonoid content

  • Perform RNA-seq to identify differentially regulated genes

  • Assess phenotypic differences, especially under stress conditions

What methodological approaches can accurately evaluate BHLH80's binding specificity to different E-box variants?

To characterize BHLH80's E-box binding preferences:

• In vitro binding assays:

  • Electrophoretic Mobility Shift Assay (EMSA) with labeled oligonucleotides containing different E-box variants

  • Surface Plasmon Resonance (SPR) to measure binding kinetics and affinity constants

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to determine optimal binding sequences

• Genome-wide binding analysis:

  • ChIP-seq using BHLH80 antibody

  • Motif enrichment analysis of binding sites

  • De novo motif discovery to identify extended binding preferences beyond the core E-box

• Structural considerations:

  • bHLH factors make specific contacts with DNA through the basic domain

  • Residues at positions 1, 2, 5, 6, 8, 9, 12 and 13 determine base-specific interactions

  • Analysis should consider how BHLH80's specific amino acid composition at these positions influences binding preference

• Competitive binding experiments:

  • Compare binding affinity to different E-box variants in competitive assays

  • Assess impact of flanking sequences on binding affinity

  • Determine effect of DNA methylation on binding efficiency

How does BHLH80 integrate with other transcription factors to form functional regulatory complexes?

Based on research with EbbHLH80 and other bHLH factors:

• MBW complex formation:

  • bHLH factors typically form part of the MYB-bHLH-WD40 (MBW) complex

  • This complex directly binds to promoters of flavonoid biosynthesis genes

  • Research suggests "MYB alone, coexpression of MYB and bHLH, or the MBW complex is sufficient to induce flavonoid accumulation in plants"

• Interaction with ERF factors:

  • EbbHLH80 overexpression led to upregulation of 49 ERF transcription factors

  • ERFs have been implicated in anthocyanin biosynthesis

  • The ethylene response factor MdERF109 promotes coloration by directly binding to promoters of anthocyanin-related genes

  • This suggests a potential coordinate regulation mechanism

• Methodological approaches to study complexes:

  • Co-immunoprecipitation with BHLH80 antibody followed by mass spectrometry

  • Yeast two-hybrid screening to identify direct interaction partners

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in planta

  • Sequential ChIP (re-ChIP) to identify co-occupancy of binding sites

• Functional consequences:

  • Different combinations of transcription factors may target distinct sets of genes

  • Interactions may be tissue-specific or condition-dependent

  • Post-translational modifications may alter complex formation and activity

What are common issues encountered with BHLH80 antibody in immunoprecipitation experiments and how can they be resolved?

Common challenges and solutions in BHLH80 immunoprecipitation:

• Low immunoprecipitation efficiency:

  • Problem: Weak or no detection of BHLH80 in IP samples

  • Solutions:

    • Increase antibody amount (5-10 μg per reaction)

    • Extend incubation time (overnight at 4°C)

    • Optimize lysis conditions to ensure complete nuclear protein extraction

    • Use crosslinking agents (formaldehyde, DSP) to stabilize protein-protein interactions

• High background or non-specific binding:

  • Problem: Multiple non-specific bands in Western blot of IP samples

  • Solutions:

    • Increase washing stringency (higher salt concentration, more detergent)

    • Pre-clear lysate with Protein A/G beads before adding antibody

    • Use more specific elution conditions

    • Consider using magnetic beads instead of agarose for cleaner results

• Antibody heavy chain interference:

  • Problem: Antibody heavy chain (~50 kDa) masks detection of similarly sized proteins

  • Solutions:

    • Use HRP-conjugated protein A/G for detection instead of secondary antibody

    • Use light-chain specific secondary antibodies

    • Consider crosslinking antibody to beads before IP

• Protein complex disruption:

  • Problem: Failure to capture protein interaction partners

  • Solutions:

    • Use gentler lysis conditions (reduce detergent concentration)

    • Add protein crosslinkers before cell lysis

    • Include phosphatase and protease inhibitors to preserve modifications

    • Optimize salt concentration to maintain complex integrity

How can researchers validate the specificity of their BHLH80 antibody results?

To ensure BHLH80 antibody specificity:

• Essential controls:

  • Positive control: Extract from tissues with known BHLH80 expression (leaf tissue shows highest EbbHLH80 expression )

  • Negative control: Extract from BHLH80 knockout/knockdown plants

  • Overexpression control: Extract from BHLH80 overexpression lines

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of closely related bHLH family members

  • Compare binding patterns across multiple plant species

  • Perform Western blot in tissues with known differential expression patterns

• Application-specific validation:

  • For Western blot: Verify single band at expected molecular weight (~45-50 kDa)

  • For ChIP: Confirm enrichment at known E-box containing promoters

  • For immunofluorescence: Include peptide competition controls

  • For IP-MS: Verify peptide coverage of BHLH80 sequence by mass spectrometry

• Orthogonal validation:

  • Compare results with a second antibody targeting a different epitope

  • Correlate protein detection with mRNA expression data

  • Validate functional findings using genetic approaches (overexpression, knockout)

What experimental design considerations are critical when studying BHLH80's role in transcriptional networks during plant stress responses?

Based on EbbHLH80 research showing involvement in stress responses :

• Stress treatment optimization:

  • Treatment types: Test multiple stress conditions (drought, salt, heat, cold, pathogen)

  • Time course: Sample at multiple timepoints (early, middle, late responses)

  • Severity gradient: Apply different intensities of stress

  • Combination stresses: Evaluate BHLH80 response under combined stresses

• Tissue-specific considerations:

  • BHLH80 expression is highest in leaves of E. breviscapus

  • Compare responses across different tissues (roots, stems, leaves)

  • Consider developmental stage effects on stress response

• Multi-omics integration:

  • Time-resolved transcriptomics: Capture dynamic gene expression changes

  • ChIP-seq: Identify direct BHLH80 targets under stress conditions

  • Metabolomics: Quantify changes in flavonoids and other metabolites

  • Proteomics: Assess post-translational modifications of BHLH80 during stress

• Comparative analysis framework:

  • Compare BHLH80 knockout and overexpression lines under stress

  • Construct transcriptional network models identifying:

    • Direct BHLH80 targets

    • Indirect regulatory effects

    • Co-regulated genes

  • Establish temporal sequence of transcriptional events