BHLH106 Antibody

What is bHLH106 and why are antibodies against it important for plant stress research?

bHLH106 is a basic helix-loop-helix transcription factor identified in Arabidopsis thaliana that plays a crucial role in salt tolerance and abiotic stress responses. The protein operates by binding to G-box sequences (5'-CACGTG-3') in the promoter regions of target genes . Antibodies against bHLH106 are essential research tools because they enable:

• Verification of protein expression in transgenic or knockout lines

• Determination of subcellular localization (primarily nuclear)

• Assessment of protein-protein interactions in stress response pathways

• Chromatin immunoprecipitation studies to identify DNA binding sites
  Research has shown that bHLH106-knockout lines exhibit increased sensitivity to NaCl, KCl, LiCl, ABA, and low temperatures compared to wild-type plants, while overexpression confers enhanced salt tolerance .

What techniques can bHLH106 antibodies be used for in plant molecular biology research?

bHLH106 antibodies can be employed in numerous experimental techniques:

How can I validate the specificity of a commercially available bHLH106 antibody?

Validating antibody specificity is crucial for reliable experimental results. For bHLH106 antibodies, implement these methodological approaches:

• Positive control testing: Use protein extracts from plants overexpressing bHLH106 alongside wild-type controls.

• Negative control validation: Include protein extracts from bHLH106-knockout lines (such as the stc8 knockout mutants) .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify that signal disappearance occurs.

• Cross-reactivity assessment: Test the antibody against other bHLH family members, particularly those with high sequence homology.

• Multiple technique confirmation: Validate protein detection across different methods (Western blot, immunoprecipitation, immunofluorescence).
  The high conservation among bHLH domains makes careful validation particularly important to ensure specificity against bHLH106 rather than related family members .

How can I optimize ChIP protocols using bHLH106 antibodies to identify novel G-box targets in stress response pathways?

Optimizing ChIP protocols for bHLH106 requires careful consideration of several factors:

• Crosslinking optimization: For bHLH transcription factors like bHLH106, a dual crosslinking approach using both formaldehyde (1% for 10 minutes) and disuccinimidyl glutarate (DSG, 2mM for 45 minutes) can improve DNA-protein fixation efficiency.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500bp, which is ideal for resolving binding sites at G-box elements (5'-CACGTG-3') and related E-box sequences (5'-CANNTG-3') .

• Antibody selection and validation:

  • Use ChIP-grade antibodies specifically validated for bHLH106

  • Perform preliminary ChIP-qPCR against known targets before proceeding to genome-wide analysis

  • Consider using epitope-tagged bHLH106 (e.g., HA or FLAG) if native antibodies show limited specificity

• Controls implementation:

  • Input chromatin (pre-immunoprecipitation)

  • IgG negative control

  • Known target regions (positive control)

  • bHLH106 knockout plants (negative control)
    For data analysis, focus on promoter regions containing G-box elements, as research has demonstrated that bHLH106 exhibits strongest interaction with these sequences compared to other E-box variants .

What approaches can resolve contradictory results when studying bHLH106 phosphorylation states using antibodies?

Resolving contradictory results regarding bHLH106 phosphorylation requires systematic troubleshooting:

• Phosphorylation-specific antibodies: Consider developing phospho-specific antibodies targeting predicted phosphorylation sites in bHLH106. Studies on related bHLH transcription factors suggest that phosphorylation can significantly alter DNA binding affinity and protein-protein interactions .

• Sample preparation considerations:

  • Include phosphatase inhibitors in all extraction buffers

  • Compare multiple protein extraction protocols

  • Perform lambda phosphatase treatment as a control

• Technical verification approaches:

  • Use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Combine immunoprecipitation with mass spectrometry

  • Employ 2D gel electrophoresis to separate isoforms

• Functional validation methods:

  • Generate phosphomimetic and phospho-null mutations of predicted sites

  • Assess binding affinity to G-box elements using EMSA with modified proteins

  • Compare DNA binding profiles of different phosphorylation states using in vitro assays
    Recent research on CDPK interactions with bHLH transcription factors suggests that phosphorylation may be a key regulatory mechanism for bHLH106 activity under stress conditions, particularly in response to salt stress .

How can I develop a co-immunoprecipitation protocol to investigate protein interactions between bHLH106 and potential CDPK partners?

Developing an effective co-immunoprecipitation (co-IP) protocol for studying bHLH106 interactions with CDPKs requires several specialized considerations:

• Protein extraction optimization:

  • Use a buffer system containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 5% glycerol, and protease inhibitors

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

  • For plant tissues, consider adding 5 mM DTT and 2% PVPP to reduce interference from phenolic compounds

• Co-IP strategy selection:

  • Antibody-based approach: Use anti-bHLH106 antibodies coupled to protein A/G beads

  • Tag-based approach: Generate plants expressing epitope-tagged bHLH106 (FLAG, HA, or Myc)

  • GST-pulldown verification: As demonstrated in CgbHLH001-CDPK interaction studies, immobilize His-tagged bHLH106 on Ni columns to pull down GST-CDPK fusion proteins

• Interaction validation:

  • Perform reciprocal co-IPs (pull down with anti-CDPK antibodies)

  • Include appropriate controls (IgG, unrelated proteins)

  • Consider BiFC assays as performed for CgbHLH001-CDPK interactions

• Sample preparation for interaction analysis:

  • Apply stress treatments (salt, ABA, cold) before extraction to capture condition-specific interactions

  • Include time course sampling to capture dynamic interaction changes
    Research on related bHLH proteins suggests that interaction with CDPK likely occurs at the helix-loop-helix domain of bHLH106 and may involve the protein kinase domain or N-variable terminal domain of CDPK .

What methodological approaches can distinguish between direct and indirect transcriptional regulation by bHLH106 when performing ChIP-seq analysis?

Distinguishing direct from indirect transcriptional regulation by bHLH106 requires a multi-faceted approach:

• Integrative genomic analysis:

  • Combine ChIP-seq data with RNA-seq from bHLH106 overexpression and knockout lines

  • Focus on genes with both bHLH106 binding sites and differential expression

  • Apply motif enrichment analysis to identify canonical G-box elements (5'-CACGTG-3') and variants

• Time-course experiments:

  • Implement rapid induction systems (e.g., dexamethasone-inducible bHLH106)

  • Early response genes (0-2 hours post-induction) are more likely direct targets

  • Late response genes may represent indirect regulation

• EMSA validation:

  • Perform electrophoretic mobility shift assays with purified bHLH106 protein

  • Test binding to putative G-box elements identified in ChIP-seq

  • Include competition assays with unlabeled probes to confirm specificity

• Reporter gene assays:

  • Construct reporters with native promoters containing bHLH106 binding sites

  • Create variants with mutated G-box elements

  • Test activation/repression in transient expression systems
    Previous studies have identified 198 genes positively regulated and 36 genes negatively regulated by bHLH106, all containing one or more G-box sequences in their promoter regions .

How can I address non-specific binding issues when using bHLH106 antibodies in plant protein extracts?

Non-specific binding is a common challenge when using antibodies against transcription factors like bHLH106. Implement these methodological solutions:

• Extraction buffer optimization:

  • Increase salt concentration (250-300 mM NaCl) to reduce ionic interactions

  • Add 0.1-0.5% SDS for Western blotting applications

  • Include 0.5% BSA or 5% non-fat dry milk as blocking agents

• Antibody dilution optimization:

  • Test multiple dilution series (1:500 to 1:5000)

  • Perform dot blot analysis to determine optimal concentration

  • Consider longer incubation at 4°C with more dilute antibody

• Pre-adsorption techniques:

  • Pre-incubate antibody with protein extract from bHLH106 knockout plants

  • Use acetone-powdered plant tissue for pre-clearing

• Alternative detection strategies:

  • Consider using epitope-tagged bHLH106 constructs

  • In ChIP applications, implement sequential ChIP (re-ChIP) to increase specificity

  • Use competition with excessive immunizing peptide as a control
    The plant bHLH family contains approximately 160 members in tomato and 116 in Erigeron breviscapus , making specificity particularly challenging due to conserved domains.

What factors should be considered when designing antibodies against specific domains of bHLH106 for functional studies?

Designing domain-specific antibodies for bHLH106 requires strategic consideration of protein structure and function:

• Domain selection considerations:

  • Basic region: Contains DNA-binding residues (crucial for G-box interaction)

  • Helix-loop-helix domain: Critical for dimerization

  • N-terminal region: May contain regulatory domains

  • C-terminal region: Often contains transactivation domains

• Epitope selection criteria:

  • Avoid highly conserved regions shared with other bHLH proteins

  • Select regions with high predicted antigenicity

  • Target sequences unique to bHLH106 versus other family members

  • Consider accessibility based on protein structure predictions

• Application-specific design:

  • For ChIP applications: Target regions not involved in DNA binding

  • For interaction studies: Avoid epitopes at protein-protein interaction interfaces

  • For phosphorylation studies: Consider phospho-specific antibodies against predicted sites

• Validation strategy:

  • Test against recombinant full-length and truncated bHLH106 proteins

  • Verify lack of cross-reactivity with related bHLH family members

  • Confirm functionality in multiple applications (Western, IP, ChIP)
    Research has shown that the bHLH domain of bHLH106 contains a conserved motif structure, with motifs 1 and 2 present in 86% of bHLH proteins , suggesting that targeting unique regions outside this domain may improve specificity.

How can bHLH106 antibodies be used to study dynamic changes in transcription factor binding during progressive salt stress?

Studying dynamic changes in bHLH106 binding during progressive salt stress requires specialized experimental approaches:

• Time-course ChIP-seq experimental design:

  • Sample at multiple time points during stress application (0, 0.5, 1, 3, 6, 12, 24 hours)

  • Apply graduated stress levels (50, 100, 150, 200 mM NaCl)

  • Include recovery phase sampling to capture resilience mechanisms

• Integrated multi-omics strategy:

  • Combine ChIP-seq, RNA-seq, and protein analysis at each time point

  • Correlate binding dynamics with transcriptional changes

  • Include protein modifications analysis (phosphorylation, ubiquitination)

• Single-cell applications:

  • Consider CUT&Tag or CUT&RUN techniques for improved sensitivity

  • Apply to specific cell types (root cells vs. leaf cells)

• Data analysis approaches:

  • Implement differential binding analysis between time points

  • Identify transient vs. sustained binding events

  • Correlate binding strength with gene expression changes

  • Apply motif enrichment analysis to identify context-specific cofactors
    Research has demonstrated that bHLH106 regulates diverse groups of genes related to ABA, ethylene, jasmonic acid, ion transport, and protein phosphorylation/dephosphorylation under salt stress conditions , suggesting dynamic regulatory mechanisms.

What methodological approaches can distinguish the roles of bHLH106 phosphorylation versus protein level changes in stress response regulation?

Distinguishing between phosphorylation effects and protein abundance changes requires sophisticated experimental design:

• Protein modification-specific methods:

  • Develop phospho-specific antibodies against predicted bHLH106 phosphorylation sites

  • Use Phos-tag gel electrophoresis to separate phosphorylated forms

  • Apply targeted mass spectrometry to quantify specific phosphopeptides

  • Implement proximity ligation assays to detect specific phosphorylated forms in situ

• Genetic complementation strategy:

  • Generate transgenic plants expressing phosphomimetic (S/T→D/E) and phospho-null (S/T→A) variants

  • Express these variants in bHLH106 knockout background

  • Compare phenotypic rescue and transcriptional profiles

  • Assess DNA binding capabilities of modified proteins

• Temporal resolution approaches:

  • Perform high-resolution time course experiments following stress application

  • Compare kinetics of phosphorylation events versus protein abundance changes

  • Use cycloheximide to block new protein synthesis and isolate post-translational effects
    Research on bHLH transcription factors in other plants suggests that CDPK-mediated phosphorylation can significantly alter DNA binding affinity and subcellular localization , providing a model for bHLH106 regulation.

How can I develop a ChIP-seq protocol specific for detecting bHLH106 heterodimers versus homodimers at G-box elements?

Developing ChIP protocols to distinguish between bHLH106 heterodimers and homodimers requires specialized techniques:

• Sequential ChIP approach:

  • Perform first ChIP with anti-bHLH106 antibodies

  • Elute complexes and perform second ChIP with antibodies against potential partners

  • Positive signal indicates heterodimer complexes

  • Include appropriate controls (IgG, unrelated TFs)

• Dual tagging strategy:

  • Generate plants expressing differentially tagged bHLH106 variants (e.g., HA-bHLH106 and FLAG-bHLH106)

  • Perform sequential ChIP with anti-HA followed by anti-FLAG

  • Enrichment indicates homodimer binding

  • Include heterodimer partners with distinct tags for comparison

• Biophysical validation approaches:

  • Combine ChIP results with in vitro DNA binding assays

  • Use size exclusion chromatography to separate complexes

  • Apply native gel electrophoresis to resolve different complexes

  • Consider atomic force microscopy to visualize complex architecture

• Motif analysis refinement:

  • Analyze spacing and orientation of G-box elements at binding sites

  • Compare motif enrichment patterns between putative homodimer and heterodimer sites

  • Assess co-occurrence of binding motifs for potential partner TFs
    Research has shown that G-box elements (5'-CACGTG-3') have the strongest interaction with bHLH106 , and understanding the dimerization patterns at these sites would provide deeper insights into regulatory mechanisms.

How can bHLH106 antibodies facilitate the study of stress memory mechanisms in plants?

bHLH106 antibodies can significantly advance our understanding of plant stress memory through these methodological approaches:

• Chromatin state analysis:

  • Perform ChIP-seq for bHLH106 binding before stress, during stress, and post-recovery

  • Correlate with histone modification patterns (H3K4me3, H3K27me3)

  • Identify persistent binding sites that maintain altered chromatin states

  • Compare binding profiles between naive plants and those with prior stress exposure

• Protein complex dynamics investigation:

  • Use serial immunoprecipitation to capture temporal changes in bHLH106-associated proteins

  • Identify stress-specific interaction partners

  • Assess post-translational modification patterns during recovery phases

  • Compare complex composition between first and subsequent stress exposures

• Transgenerational analysis:

  • Apply bHLH106 ChIP-seq to successive plant generations following parental stress

  • Correlate with DNA methylation patterns in offspring

  • Assess heritability of binding patterns and associated gene expression changes
    Research has shown that bHLH106 overexpression confers salt tolerance in plants , suggesting it may be involved in establishing persistent stress adaptation mechanisms.

What cutting-edge approaches can combine bHLH106 antibodies with CRISPR technologies to elucidate its role in stress response networks?

Integrating bHLH106 antibodies with CRISPR technologies enables sophisticated functional studies:

• CRISPRi/CRISPRa applications:

  • Use dCas9-based approaches to modulate bHLH106 expression in specific tissues

  • Apply bHLH106 antibodies to confirm altered protein levels

  • Perform ChIP-seq to assess binding pattern changes

  • Correlate with transcriptome alterations

• Base editing techniques:

  • Generate precise mutations in bHLH106 DNA-binding domain or dimerization interfaces

  • Use antibodies to confirm protein stability and localization

  • Assess altered binding profiles via ChIP-seq

  • Identify functionally critical residues for specific stress responses

• CRISPR screening applications:

  • Develop CRISPR screens targeting G-box elements in bHLH106 target promoters

  • Use antibodies to assess changes in bHLH106 recruitment

  • Identify essential target genes in stress response networks

  • Conduct epistasis analysis with bHLH106 mutants

• Temporal control strategies:

  • Implement optogenetic or chemically inducible CRISPR systems to control bHLH106 activity

  • Use antibodies to validate system functionality

  • Profile binding dynamics during controlled activation/inactivation

  • Determine minimal induction requirements for stress adaptation
    Recent studies have identified 198 genes positively regulated and 36 genes negatively regulated by bHLH106 through G-box elements , providing a foundation for targeted CRISPR approaches.