BHLH153 Antibody

What is BHLH153 and what is its role in plant biology?

BHLH153 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. In Arabidopsis thaliana, this protein functions as a transcription factor involved in regulatory networks affecting plant development and environmental responses . The bHLH domain contains approximately 60 amino acids with two functionally distinct regions: the basic region that functions as a DNA-binding motif, and the HLH region containing two amphipathic α-helices with a linking loop that facilitates protein dimerization . BHLH153, like other members of the bHLH family, likely binds to E-box (5'-CANNTG-3') or G-box (5'-CACGTG-3') motifs in promoter regions of target genes .

What applications can BHLH153 antibodies be used for in plant research?

BHLH153 antibodies can be employed for multiple research applications:

Similar to other plant bHLH antibodies, BHLH153 antibodies can help researchers study protein expression patterns during development or in response to environmental stimuli .

How can I validate the specificity of a BHLH153 antibody?

Proper validation of BHLH153 antibodies is critical to ensure experimental rigor:

• Knockout/Knockdown Controls: Use tissue from bhlh153 knockout/knockdown plants as negative controls in your experiments

• Recombinant Protein Controls: Test antibody against purified recombinant BHLH153 protein

• Peptide Competition Assay: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Multiple Antibody Validation: Use antibodies targeting different epitopes of BHLH153

• Cross-Reactivity Testing: Test against closely related bHLH family members to assess specificity

As demonstrated in Arabidopsis antibody resources, affinity purification of antibodies massively improves detection rates, with purified antibodies showing 55% detection success compared to much lower rates for unpurified antibodies .

What factors should I consider when designing experiments using BHLH153 antibody for immunolocalization?

When designing immunolocalization experiments:

• Fixation Method: For plant tissues, PFA fixation (typically 4%) is recommended for preserving protein epitopes while maintaining cellular architecture

• Tissue Processing: Consider using fresh-frozen sections (10 μm) for better epitope preservation compared to paraffin embedding

• Antigen Retrieval: May be necessary for formalin-fixed tissues; test both heat-induced and enzymatic methods

• Blocking Conditions: Use 5-10% normal serum from the species of secondary antibody plus 0.1-0.3% Triton X-100

• Antibody Dilutions: Perform titration experiments (typically 1:50-1:500) to determine optimal concentration

• Incubation Times: Primary antibody incubation for 90 minutes at room temperature or overnight at 4°C has shown good results for similar bHLH antibodies

• Controls: Include no-primary antibody controls and, if possible, tissue from bhlh153 mutant plants

For visualization, fluorescent-conjugated secondary antibodies with nuclear counterstain (e.g., Hoechst) allow for precise subcellular localization analysis, as demonstrated with other bHLH protein antibodies .

How can I optimize Western blot protocols for BHLH153 detection in plant samples?

For optimal Western blot detection of BHLH153:

• Sample Preparation:

  • Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitors

  • Include reducing agents (DTT or β-mercaptoethanol) to break disulfide bonds

  • Consider tissue-specific extraction protocols as protein abundance may vary

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of BHLH153 (~25-30 kDa range)

  • Load sufficient protein (30-50 μg total protein per lane)

• Transfer Conditions:

  • Semi-dry transfer: 15V for 30 minutes or wet transfer: 100V for 1 hour

  • Use PVDF membranes for higher protein binding capacity

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk in TBST

  • Primary antibody dilution typically 1:1000

  • Incubate overnight at 4°C for better sensitivity

• Detection Method:

  • Use HRP-conjugated secondary antibody with enhanced chemiluminescence detection

  • Consider using fluorescently-labeled secondary antibodies for multiplex detection

• Controls:

  • Include recombinant BHLH153 as positive control

  • Use plant samples with known expression levels as reference

  • Include samples from bhlh153 mutant plants as negative control

What are the best approaches for ChIP experiments using BHLH153 antibody?

For effective ChIP experiments with BHLH153 antibody:

• Crosslinking and Chromatin Preparation:

  • Use 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125 M glycine

  • Optimize sonication to achieve DNA fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5 μg antibody per reaction

  • Include IgG control and input samples

  • Incubate overnight at 4°C with rotation

• Washing and Elution:

  • Use stringent wash buffers to reduce background

  • Elute DNA-protein complexes with elution buffer (1% SDS, 0.1 M NaHCO₃)

  • Reverse crosslinks with proteinase K treatment and heat

• DNA Analysis:

  • Use qPCR to analyze specific targets (E-box/G-box containing promoters)

  • For genome-wide analysis, perform ChIP-seq

• Data Validation:

  • Focus on regions containing the E-box (5'-CANNTG-3') or G-box (5'-CACGTG-3') motifs, as these are the canonical binding sites for bHLH proteins

  • Validate with reporter gene assays or in vitro DNA binding studies

How can I determine if BHLH153 forms heterodimers with other bHLH proteins, and how does this affect its function?

To investigate BHLH153 heterodimerization:

• Co-Immunoprecipitation (Co-IP):

  • Use BHLH153 antibody to pull down protein complexes from plant extracts

  • Analyze precipitated proteins by mass spectrometry to identify interaction partners

  • Confirm interactions with reciprocal Co-IP using antibodies against potential partners

• Yeast Two-Hybrid Screening:

  • Use BHLH153 as bait to screen for interacting proteins

  • Follow approaches similar to those used for identifying ATBS1-Interacting Factors (AIFs)

  • Validate interactions with GST pull-down assays using purified proteins

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of BHLH153 and candidate partners with split YFP fragments

  • Co-express in plant cells to visualize interactions in vivo

  • This approach has been successful for confirming interactions between other bHLH proteins

• Functional Analysis of Heterodimers:

  • Generate transgenic plants co-expressing BHLH153 and interaction partners

  • Compare phenotypes with single overexpression lines

  • Perform ChIP-seq analyses to identify differential DNA binding sites

The antagonistic relationship between bHLH proteins can be critical for their function, as demonstrated by the interaction between ILI1 and IBH1, or PRE1 and AtIBH1, which form pairs of antagonistic HLH/bHLH transcription factors mediating brassinosteroid regulation of cell elongation .

What role does phosphorylation play in regulating BHLH153 activity, and how can I study this?

Investigating BHLH153 phosphorylation:

• Identification of Phosphorylation Sites:

  • Perform mass spectrometry analysis of immunoprecipitated BHLH153

  • Compare phosphorylation patterns under different conditions/treatments

  • Generate phospho-specific antibodies for key sites

• Kinase Identification:

  • Use in vitro kinase assays with candidate kinases (e.g., MPK3/6)

  • Perform Co-IP experiments to identify associated kinases

  • Recent research has shown that MPK3/6-mediated phosphorylation can regulate bHLH transcription factors like ICE1/SCRM in different developmental contexts

• Functional Analysis of Phosphorylation:

  • Generate phospho-mimetic (S/T to D/E) and phospho-null (S/T to A) mutants

  • Compare DNA binding, protein stability, and subcellular localization

  • Assess transcriptional activity using reporter gene assays

• Biological Significance:

  • Express phospho-variants in bhlh153 mutant background

  • Analyze phenotypic consequences

  • Perform ChIP-seq to identify differential target binding

As demonstrated with the bHLH transcription factor ICE1/SCRM, phosphorylation can have context-dependent effects, either activating or inhibiting transcription factor function depending on the cellular context .

How does BHLH153 contribute to the transcriptional regulation network in response to environmental stress?

To investigate BHLH153's role in stress response networks:

• Transcriptome Analysis:

  • Compare RNA-seq data between wild-type and bhlh153 mutant plants under various stress conditions

  • Identify differentially expressed genes, focusing on those containing E-box or G-box motifs

  • Perform Gene Ontology enrichment analysis to identify biological processes affected

• ChIP-seq Analysis:

  • Map genome-wide binding sites of BHLH153 under normal and stress conditions

  • Integrate with transcriptome data to identify direct targets

  • Analyze binding site motifs to determine if stress alters binding preferences

• Protein-Protein Interaction Networks:

  • Identify changes in BHLH153 interaction partners under stress conditions

  • Investigate interactions with other transcription factors and chromatin modifiers

  • Similar approaches have revealed how bHLH proteins like HBI1 mediate trade-offs between growth and immunity in Arabidopsis

• Functional Validation:

  • Generate transgenic lines with inducible BHLH153 expression

  • Assess phenotypic responses to various stresses

  • Perform promoter analysis of key target genes

Many bHLH proteins function in stress responses. For example, ThbHLH1 from Tamarix hispida is highly expressed under salt stress and increases activities of stress-related enzymes; AtbHLH112 enhances resistance to salt stress by regulating expression of POD and SOD genes .

What are the considerations for developing a highly specific antibody against BHLH153?

For developing specific BHLH153 antibodies:

• Epitope Selection:

  • Target unique regions outside the conserved bHLH domain to minimize cross-reactivity

  • Use bioinformatics analysis to identify BHLH153-specific sequences

  • Consider multiple epitopes for different antibody development strategies

• Immunization Strategy:

  • Options include:
    a) Synthetic peptides conjugated to carrier proteins
    b) Recombinant protein fragments
    c) Full-length recombinant protein

  • The success rate with peptide antibodies is typically lower than with recombinant protein antigens

• Host Selection:

  • Rabbit polyclonal antibodies provide good sensitivity

  • Consider monoclonal antibody development for long-term reproducibility

  • Alternative hosts (chicken, goat) may offer advantages for certain applications

• Purification Methods:

  • Affinity purification using the immunizing antigen significantly improves specificity

  • Consider negative selection against related bHLH proteins

  • Implement rigorous quality control testing

• Validation Strategy:

  • Test against recombinant BHLH153 and related bHLH proteins

  • Evaluate in tissues with known expression patterns

  • Validate in knockout/knockdown plant materials

Research on Arabidopsis antibody resources showed that of 70 protein antibodies developed, only 38 (55%) could detect a signal with high confidence, highlighting the importance of proper development and validation strategies .

How can I determine the optimal antibody concentration for different experimental applications?

To determine optimal antibody concentrations:

• Western Blotting:

  • Perform titration experiments with 1:500, 1:1000, 1:2000, and 1:5000 dilutions

  • Evaluate signal-to-noise ratio at each concentration

  • Test different blocking agents (BSA vs. milk) to optimize conditions

  • For similar bHLH antibodies, 1:1000 dilution has been effective

• Immunohistochemistry/Immunofluorescence:

  • Start with manufacturer's recommended dilution (typically 1:100-1:500)

  • Perform serial dilutions to identify optimal concentration

  • Consider testing different antigen retrieval methods

  • Antibody dilution of 1:100 has shown good results for similar bHLH antibodies

• ChIP Experiments:

  • Typically requires 2-5 μg antibody per reaction

  • Perform pilot experiments with different amounts

  • Evaluate enrichment of known targets by qPCR

• Flow Cytometry:

  • Start with 1-5 μg/mL and adjust based on results

  • Optimize fixation and permeabilization conditions

• Optimization Table Example:

What are the advantages and limitations of using phage display versus animal immunization for generating BHLH153 antibodies?

The phage display approach offers several advantages for generating antibodies against conserved plant proteins like BHLH153:

• The ability to select against specific epitopes allows targeting unique regions outside the conserved bHLH domain

• Negative selection against related bHLH proteins can dramatically improve specificity

• The absence of immunological tolerance makes it possible to develop antibodies against highly conserved proteins

As demonstrated in search result , phage-displayed human naive scFv and Fab libraries have been successfully used to identify antibodies with high binding affinity (KD = 10⁻⁹-10⁻¹⁰ M) against various target antigens, and affinity maturation strategies can further improve binding to KD of 10⁻¹⁰-10⁻¹¹ M .

What are common causes of non-specific binding when using BHLH153 antibodies, and how can these be addressed?

Common causes of non-specific binding and their solutions:

• Cross-reactivity with Related bHLH Proteins:

  • Problem: The bHLH domain is highly conserved across family members

  • Solution:

    • Use antibodies targeting unique N- or C-terminal regions

    • Perform pre-absorption with recombinant related bHLH proteins

    • Use higher dilutions of antibody

    • Include competitive peptide controls

• Insufficient Blocking:

  • Problem: Inadequate blocking allows antibody binding to non-specific sites

  • Solution:

    • Increase blocking time (1-2 hours at room temperature)

    • Try different blocking agents (BSA, milk, normal serum)

    • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

    • Consider commercial blocking solutions with proprietary formulations

• Overfixation:

  • Problem: Excessive fixation can create artificial epitopes

  • Solution:

    • Optimize fixation time and concentration

    • Test alternative fixatives

    • Implement appropriate antigen retrieval methods

• High Background in Plant Tissues:

  • Problem: Plant tissues often contain compounds that interact with antibodies

  • Solution:

    • Pre-incubate tissues with non-immune serum from secondary antibody species

    • Add 0.1% gelatin or 1% BSA to antibody dilution buffer

    • Include 0.1-0.3% Tween-20 in wash buffers

    • Consider using plant-optimized detection systems

• Experimental Protocol Optimization:

  • Increase washing steps in number and duration

  • Reduce primary antibody concentration

  • Decrease incubation temperature (4°C overnight versus room temperature)

  • Use more stringent wash buffers (higher salt concentration)

How can I address weak or absent signals when working with BHLH153 antibody?

Troubleshooting weak or absent signals:

• Protein Expression Level Issues:

  • Problem: BHLH153 may be expressed at low levels in your sample

  • Solution:

    • Enrich for nuclear proteins (BHLH153 is a transcription factor)

    • Use tissues/conditions known to express BHLH153

    • Consider using an overexpression system for positive controls

• Epitope Accessibility Problems:

  • Problem: Epitope may be masked due to protein folding or interactions

  • Solution:

    • Try different sample preparation methods (native vs. denaturing)

    • Implement antigen retrieval techniques for fixed tissues

    • Test different fixation methods for immunohistochemistry

    • Consider using multiple antibodies targeting different epitopes

• Protocol Optimization:

  • Increase antibody concentration or incubation time

  • Optimize detection system (try more sensitive ECL reagents for Western blot)

  • Reduce washing stringency

  • For IHC/IF, try signal amplification methods (tyramide signal amplification)

• Antibody Storage and Handling:

  • Avoid freeze-thaw cycles

  • Store antibody according to manufacturer's recommendations

  • Add preservatives (0.02% sodium azide) for long-term storage

  • Aliquot stock antibody to prevent degradation

• Sample Preparation Considerations:

  • Include protease inhibitors in extraction buffers

  • Add phosphatase inhibitors if phosphorylation is important

  • Maintain cold chain during sample preparation

  • Consider using fresh samples rather than frozen

As demonstrated in other bHLH antibody applications, affinity purification of antibodies significantly improves detection success rates .

What alternative approaches can I use to study BHLH153 expression and function if antibody-based methods are unsuccessful?

When antibody-based methods prove challenging, consider these alternatives:

• Epitope Tagging Approaches:

  • Generate transgenic plants expressing BHLH153 with epitope tags (HA, FLAG, Myc)

  • Use well-characterized commercial antibodies against these tags

  • Create GFP/YFP fusion proteins for direct visualization

  • This approach has been successful for other bHLH proteins, as demonstrated with ATBS1-GFP

• Transcript Analysis Methods:

  • RT-qPCR for quantitative expression analysis

  • RNA in situ hybridization for spatial expression patterns

  • Single-cell RNA-seq for cell-type specific expression

  • RNA-seq for genome-wide expression analysis and identification of downstream genes

• Promoter-Reporter Systems:

  • Generate pBHLH153::GUS or pBHLH153::GFP constructs

  • Analyze reporter expression patterns in various tissues/conditions

  • Use for promoter deletion analysis to identify regulatory elements

• CRISPR-Based Approaches:

  • CRISPR/Cas9 for gene knockout studies

  • CRISPRa/CRISPRi for modulating gene expression

  • CRISPR-based genomic tagging (e.g., CRISPR knock-in of tags)

  • Recent studies have utilized CRISPR/Cas9 to generate loss-of-function alleles in bHLH genes

• Chromatin Analysis Techniques:

  • ATAC-seq to identify open chromatin regions

  • DNase-seq for accessible DNA identification

  • Chromosome conformation capture (3C, 4C, Hi-C) for chromatin interactions

• Yeast-Based Systems:

  • Yeast one-hybrid for DNA-binding studies

  • Yeast two-hybrid for protein interaction screening

  • This approach has been successful in identifying interaction partners for bHLH proteins

These methodologies provide complementary information to antibody-based techniques and may overcome limitations associated with antibody availability or specificity.

How can new antibody technologies enhance BHLH153 research?

Emerging antibody technologies with potential impact on BHLH153 research:

• Single B Cell Technologies:

  • Enables rapid production of monoclonal antibodies

  • Collects antigen-specific memory B cells by flow cytometry

  • Directly obtains antibody sequences through RT-PCR

  • Can produce antibodies in less than a month, compared to traditional methods

• Nanobodies/Single-Domain Antibodies:

  • Derived from camelid heavy-chain antibodies

  • Smaller size (~15 kDa vs. ~150 kDa for conventional antibodies)

  • Better penetration into tissues and cells

  • Higher stability under varying conditions

  • Potential for improved detection of transcription factors in fixed tissues

• Proximity-Dependent Labeling:

  • Antibody-enzyme fusions (e.g., APEX2, BioID, TurboID)

  • Label proteins in proximity to BHLH153 in living cells

  • Map protein interaction networks in native context

  • Identify transient interactions difficult to capture by Co-IP

• Multiplexed Antibody Detection Systems:

  • Simultaneous detection of multiple bHLH family members

  • Co-localization studies with downstream targets

  • Signal amplification methods for low-abundance targets

  • Mass cytometry (CyTOF) for high-dimensional analysis

• Engineered Antibody Fragments:

  • scFv (single-chain variable fragments)

  • Fab fragments

  • Bispecific antibodies targeting BHLH153 and interacting partners

  • As demonstrated in the search results, bispecific antibodies offer advantages in terms of biosuperiority over monotherapy or combination treatment with novel mechanisms of action

What are the current limitations in our understanding of BHLH153 function, and how might new antibody approaches address these?

Current knowledge gaps and potential solutions:

• Tissue-Specific Function:

  • Knowledge Gap: Limited understanding of tissue-specific roles

  • Antibody Solution: Development of highly sensitive antibodies for tissue-specific immunohistochemistry

  • Alternative Approach: Single-cell proteomics with BHLH153-specific antibodies

• Protein-Protein Interaction Networks:

  • Knowledge Gap: Incomplete map of interaction partners

  • Antibody Solution: Proximity labeling with antibody-enzyme fusions

  • Alternative Approach: Co-IP followed by mass spectrometry using high-affinity antibodies

• Post-Translational Modifications:

  • Knowledge Gap: Limited information on regulatory modifications

  • Antibody Solution: Development of modification-specific antibodies (phospho-, acetyl-, ubiquitin-specific)

  • Alternative Approach: Mass spectrometry-based PTM mapping of immunoprecipitated BHLH153

• Dynamic Regulation:

  • Knowledge Gap: Temporal changes in BHLH153 activity

  • Antibody Solution: Live-cell imaging with engineered antibody fragments

  • Alternative Approach: Time-course studies with quantitative proteomics

• Target Gene Specificity:

  • Knowledge Gap: Incomplete understanding of DNA binding specificity

  • Antibody Solution: ChIP-seq with highly specific antibodies

  • Alternative Approach: CUT&RUN or CUT&Tag using BHLH153 antibodies for higher resolution mapping

As demonstrated by studies with other bHLH proteins, understanding the phosphorylation-dependent regulation of these transcription factors can provide critical insights into their biological functions .

How can integrative approaches combining antibody-based methods with other technologies advance our understanding of BHLH153 in plant signaling networks?

Integrative approaches for comprehensive BHLH153 research:

• Multi-omics Integration:

  • Combine ChIP-seq (antibody-based) with RNA-seq to correlate binding with expression

  • Integrate proteomics data from Co-IP experiments with transcriptomics

  • Similar approaches with other bHLH proteins have revealed their roles in regulating flavonoid biosynthesis

• Spatial and Temporal Resolution:

  • Combine single-cell RNA-seq with immunohistochemistry for spatial context

  • Implement time-resolved proteomics with antibody-based enrichment

  • Correlate protein localization changes with gene expression dynamics

• Functional Networks:

  • Map protein-protein interactions using antibody-based methods

  • Correlate with genetic interaction networks from mutant studies

  • Similar approaches have revealed how bHLH proteins like HBI1 function as nodes in growth-immunity tradeoffs

• Structural Biology Integration:

  • Use antibodies to purify native protein complexes for structural studies

  • Combine with cryo-EM for structure determination

  • Implement crosslinking mass spectrometry for interaction interfaces

• Systems Biology Modeling:

  • Use quantitative data from antibody-based assays for model parameterization

  • Generate predictive models of BHLH153 function in signaling networks

  • Test model predictions with targeted experiments

• Example Research Pipeline: