BHLH35 Antibody

What is BHLH35 and why is it important in plant research?

BHLH35 (basic helix-loop-helix 35) is a transcription factor belonging to the bHLH family that plays a significant role in regulating anthocyanin biosynthesis in plants. It is particularly important in peach fruit, where it works alongside other transcription factors to control pigmentation processes. The bHLH transcription factor family represents the second most populated family of transcription factors in the human genome, with over 100 members sharing a common structural motif in their DNA binding domain . In plants like peach, PpbHLH35 has been specifically identified as a key regulatory element that works in conjunction with MYB transcription factors to control the expression of genes involved in anthocyanin production . This makes it a valuable target for research into fruit pigmentation, plant stress responses, and genetic manipulation of valuable crop traits.

What are the common challenges in generating antibodies against BHLH35?

Generating antibodies against transcription factors like BHLH35 presents several technical challenges. First, transcription factors are often expressed at relatively low levels in cells, making native protein isolation difficult. Second, the high conservation of structural domains across the bHLH family can lead to cross-reactivity issues, where antibodies recognize related family members. Third, the basic helix-loop-helix structure may present conformational epitopes that are difficult to maintain during immunization procedures.

A robust approach to overcome these challenges involves the use of recombinant antigen expression systems, where the BHLH35 protein or specific peptide fragments can be produced in sufficient quantities. For example, research protocols have demonstrated success using HEK293 cells for expression of recombinant proteins with human IgG1 Fc fusion tags to generate immunization antigens . Additionally, peripheral B-cell isolation techniques from immunized rabbits provide a powerful method for generating highly specific monoclonal antibodies against challenging targets like transcription factors .

How can I validate the specificity of a BHLH35 antibody?

Validating antibody specificity for BHLH35 requires multiple complementary approaches:

• Western blot analysis: Compare samples from tissues/cells known to express BHLH35 with negative controls (knockouts or tissues not expressing the protein).

• Immunoprecipitation followed by mass spectrometry: This confirms that the antibody captures the intended protein.

• Cross-reactivity testing: Test against related bHLH family members to ensure specificity, especially important given that the bHLH family shares a common structural motif of an alpha-helix with a basic domain followed by a loop and a second alpha-helix .

• Epitope mapping: Determine which specific region of BHLH35 the antibody recognizes using peptide arrays or competition assays similar to the epitope grouping by cross-competition ELISA techniques described in antibody characterization literature .

• Chromatin immunoprecipitation (ChIP): Verify that the antibody can detect BHLH35 bound to its target DNA sequences, which typically include the CANNTG motif (E-box) recognized by bHLH transcription factors .

An effective validation strategy should include positive controls where the antibody is tested against recombinant BHLH35 protein and negative controls using tissues or cells where BHLH35 expression has been knocked down or is naturally absent.

What sample preparation methods work best for BHLH35 antibody applications?

For optimal detection of BHLH35 using antibodies, sample preparation should account for the nuclear localization and DNA-binding properties of this transcription factor:

Nuclear Extraction Protocol:

• Harvest fresh plant tissue (e.g., peach fruit flesh for PpbHLH35) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining freezing temperatures

• Extract with nuclear isolation buffer (typically containing 20 mM HEPES pH 7.4, 10 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, and protease inhibitors)

• Separate nuclei by differential centrifugation

• Extract nuclear proteins using high-salt buffer (typically 20 mM HEPES pH 7.4, 400 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, and protease inhibitors)

For immunoprecipitation applications, gentle lysis conditions that preserve protein-protein interactions are essential, particularly when studying BHLH35's interactions with partner proteins like MYB transcription factors, which have been shown to form functional complexes in anthocyanin regulation pathways . For western blot applications, standard SDS-PAGE conditions can be used, but transfer conditions should be optimized for the molecular weight of BHLH35 and any fusion tags or post-translational modifications present.

How do I optimize ChIP protocols for BHLH35 antibodies?

Optimizing Chromatin Immunoprecipitation (ChIP) protocols for BHLH35 antibodies requires special considerations due to the specific DNA-binding properties of this transcription factor:

Optimized ChIP Protocol for BHLH35:

• Crosslinking optimization: Test multiple formaldehyde concentrations (0.5-2%) and incubation times (5-20 minutes) to determine optimal conditions for BHLH35-DNA complexes. bHLH factors recognize a specific CANNTG motif (E-box) , so crosslinking efficiency is crucial.

• Sonication parameters: Adjust sonication to achieve chromatin fragments of 200-500 bp, which is optimal for capturing BHLH35 binding sites.

• Antibody titration: Perform a titration series (2-10 μg per reaction) to determine the minimum amount of antibody needed for efficient immunoprecipitation while minimizing background.

• Pre-clearing strategy: Include a thorough pre-clearing step using protein A/G beads and non-specific IgG to reduce background binding.

• Washing stringency: Implement a progressively stringent washing series to remove non-specific interactions while preserving genuine BHLH35-DNA complexes.

• Control selection: Include both negative controls (IgG, non-expressing tissue) and positive controls (known BHLH35 target genes like UFGT in the case of PpbHLH35 ).

The optimal protocol should be validated by qPCR of known target regions before proceeding to genome-wide analyses like ChIP-seq, focusing on regions containing the E-box motif which is recognized by bHLH transcription factors.

What are the best approaches for studying BHLH35 interactions with other transcription factors?

BHLH35 functions within a complex regulatory network, particularly with MYB transcription factors to form the MYB-bHLH-WD40 (MBW) complex for anthocyanin regulation. The following methodologies are particularly effective for studying these interactions:

Co-immunoprecipitation (Co-IP) Strategy:

• Perform reciprocal Co-IPs using antibodies against both BHLH35 and suspected partner proteins (e.g., MYB44-like in peach )

• Include appropriate controls (IgG, lysates from tissues not expressing one partner)

• Verify interactions by western blot or mass spectrometry

Bimolecular Fluorescence Complementation (BiFC):

• Generate fusion constructs of BHLH35 and potential partners with split fluorescent protein fragments

• Co-express in plant protoplasts or through transient expression systems

• Visualize reconstituted fluorescence indicating protein-protein interactions

• Include appropriate negative controls with mutated interaction domains

Dual Luciferase Reporter Assays:
Similar to the approach described in the peach anthocyanin study, where PpMYB44-like was co-infiltrated with PpbHLH35 to test activation of the PpUFGT promoter . This technique can reveal functional consequences of interactions:

• Clone promoter regions of suspected target genes upstream of a luciferase reporter

• Co-express BHLH35 with potential partner proteins

• Measure luciferase activity to quantify transcriptional activation

• Include appropriate controls (empty vectors, single transcription factor expressions)

The tobacco leaf dual luciferase assay system has proven particularly effective for studying plant transcription factor interactions, as demonstrated in the study of PpbHLH35's interaction with PpMYB44-like in activating the PpUFGT promoter .

How can BHLH35 antibodies be used to study post-translational modifications?

Post-translational modifications (PTMs) often regulate transcription factor activity, and BHLH35 is likely subject to such regulation. Here's how antibodies can be leveraged to study these modifications:

PTM-Specific Antibody Approach:

• Generate antibodies against predicted modification sites (phosphorylation, acetylation, ubiquitination)

• Validate using synthetic modified peptides

• Apply in western blot analysis comparing different cellular conditions

Immunoprecipitation-Mass Spectrometry (IP-MS) Strategy:

• Immunoprecipitate BHLH35 using validated antibodies

• Process samples for mass spectrometry analysis

• Identify PTMs through specialized MS protocols

• Quantify modification stoichiometry under different conditions

Functional Analysis of PTMs:

• Compare DNA-binding activity of modified versus unmodified BHLH35

• Assess impact on protein-protein interactions, particularly with MYB partners

• Determine effects on transcriptional activation using reporter assays

When studying PTMs, it's important to include phosphatase inhibitors (for phosphorylation studies), deacetylase inhibitors (for acetylation studies), or proteasome inhibitors (for ubiquitination studies) during sample preparation to preserve the modification state of BHLH35.

What are the critical considerations for using BHLH35 antibodies in different plant species?

When extending BHLH35 antibody applications across different plant species, researchers should consider several critical factors:

Cross-Reactivity Analysis:

Epitope Conservation Assessment:

• Perform sequence alignment of BHLH35 orthologs across target species

• Identify conserved regions, particularly in the basic helix-loop-helix domain

• Select antibodies targeting highly conserved epitopes for cross-species applications

• For variable regions, consider developing species-specific antibodies

Validation Requirements:

• Confirm specificity in each species using positive controls (recombinant protein)

• Verify using negative controls (knockout lines where available)

• Optimize immunoprecipitation conditions for each species

• Validate functional assays (e.g., ChIP) individually for each species

The structural conservation of the bHLH domain across species provides a basis for potential cross-reactivity, but careful validation is essential as even small sequence variations can affect antibody recognition.

How can I use BHLH35 antibodies to map genome-wide binding profiles?

Mapping genome-wide binding profiles of BHLH35 requires specialized approaches that leverage antibody specificity for chromatin immunoprecipitation followed by sequencing (ChIP-seq):

Optimized ChIP-seq Protocol:

• Perform crosslinking of plant tissue under optimized conditions

• Sonicate chromatin to appropriate fragment size (200-500 bp)

• Immunoprecipitate BHLH35-bound DNA using validated antibodies

• Prepare sequencing libraries from immunoprecipitated DNA

• Sequence using high-throughput platforms

• Analyze data with appropriate peak-calling algorithms

• Verify enrichment of the CANNTG (E-box) motif recognized by bHLH factors

Data Analysis Considerations:

• Use IgG or input controls for background normalization

• Apply false discovery rate controls for peak identification

• Perform motif enrichment analysis to confirm enrichment of bHLH binding sites

• Integrate with RNA-seq data to correlate binding with transcriptional outcomes

• Compare binding profiles under different conditions (e.g., sugar treatment for PpbHLH35 )

Validation Approaches:

• Confirm selected binding sites by ChIP-qPCR

• Functionally validate through reporter assays

• Compare binding profiles with known anthocyanin biosynthesis genes like UFGT

When analyzing ChIP-seq data for BHLH35, it's critical to consider its tendency to function in complexes with other transcription factors, particularly MYB proteins, as demonstrated in the regulation of anthocyanin biosynthesis in peach .

What protocols exist for using BHLH35 antibodies in tissue immunohistochemistry?

While transcription factor immunohistochemistry presents challenges due to typically low expression levels, the following optimized protocol can be effective for BHLH35 detection in plant tissues:

Tissue Preparation and Fixation:

• Harvest fresh plant tissue and immediately fix in 4% paraformaldehyde

• Process through ethanol series for dehydration

• Embed in paraffin or optimal cutting temperature compound

• Section at 5-10 μm thickness

• Mount on adhesive slides

Antigen Retrieval and Staining Protocol:

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Block with 5% normal serum from the same species as the secondary antibody

• Incubate with primary BHLH35 antibody at optimized dilution (typically 1:100 to 1:500) overnight at 4°C

• Wash extensively with PBS containing 0.1% Tween-20

• Apply fluorescently-labeled or HRP-conjugated secondary antibody

• For HRP detection, develop with DAB substrate

• Counterstain nuclei with DAPI for fluorescent detection

• Mount in appropriate medium

Controls and Validation:

• Include negative controls (primary antibody omission, pre-immune serum)

• Use positive controls (tissues known to express BHLH35)

• Consider dual staining with markers for specific cell types

• Validate patterns with in situ hybridization for BHLH35 mRNA

This approach can be particularly valuable for studying tissue-specific expression patterns of BHLH35 during fruit development or in response to sugar treatments, as observed in peach fruit anthocyanin accumulation studies .

What are common pitfalls in BHLH35 western blotting and how can they be resolved?

Western blotting for transcription factors like BHLH35 presents several technical challenges that researchers commonly encounter:

Common Problems and Solutions:

Optimization Strategies:

• For nuclear proteins like BHLH35, always include a nuclear extraction step

• Use PVDF membranes rather than nitrocellulose for better protein retention

• Optimize transfer conditions for the predicted molecular weight of BHLH35

• Consider transfer in the presence of SDS for nuclear proteins

• Validate with recombinant BHLH35 protein as a positive control

When troubleshooting, it's helpful to remember that bHLH transcription factors often form dimers , which can affect their migration pattern on gels, particularly if these interactions are not fully disrupted during sample preparation.

How do I interpret conflicting results between different BHLH35 antibody applications?

When facing discrepancies between different BHLH35 antibody applications (e.g., western blot showing positive results but ChIP showing negative results), systematic troubleshooting is required:

Methodological Reconciliation Approach:

• Epitope accessibility analysis: Different applications expose different protein regions. An epitope accessible in denatured western blotting may be masked in native ChIP applications.

• Antibody specificity verification: Confirm specificity in each application separately.

  • For western blots: Include recombinant protein controls

  • For ChIP: Validate with known target regions

  • For IP: Confirm pulled-down protein by mass spectrometry

• Protein state considerations: BHLH35 may undergo context-dependent modifications or conformational changes.

  • Compare native vs. denatured detection methods

  • Assess different cellular fractions

  • Consider stimulus-dependent modifications (e.g., sugar treatment effects on PpbHLH35 )

• Experimental condition reconciliation: Systematically adjust conditions to identify variables causing discrepancies.

  • Test multiple antibody concentrations

  • Vary fixation/extraction conditions

  • Compare results across different tissues/timepoints

Decision Tree for Resolving Conflicts:

• If western blot positive but ChIP negative: Focus on fixation conditions and epitope accessibility

• If ChIP positive but western blot negative: Consider protein abundance and extraction efficiency

• If cell staining inconsistent with biochemical methods: Evaluate fixation artifacts and antibody penetration

Understanding that BHLH35 functions in complex with other proteins, particularly MYB transcription factors , can help interpret differences between detection methods that may differentially preserve or disrupt these complexes.

How can BHLH35 antibodies be used to study the MYB-bHLH-WD40 complex in anthocyanin regulation?

The MYB-bHLH-WD40 (MBW) complex is a key regulatory mechanism in anthocyanin biosynthesis, and BHLH35 antibodies can provide valuable insights into its composition and function:

Complex Isolation Protocol:

• Cross-link plant tissue under mild conditions to preserve protein-protein interactions

• Extract nuclear proteins under native conditions

• Perform immunoprecipitation using BHLH35 antibodies

• Analyze co-precipitated proteins by mass spectrometry or western blotting

• Confirm interactions with reciprocal IPs using antibodies against MYB and WD40 partners

Functional Analysis Approaches:

• DNA-binding assessment: Use electrophoretic mobility shift assays (EMSA) with BHLH35 antibodies to supershift complexes bound to target promoters

• ChIP-reChIP: Perform sequential ChIP using first BHLH35 antibodies then MYB antibodies to identify genomic regions bound by the complete complex

• Transient expression systems: As demonstrated in the study of peach anthocyanin regulation, use systems like tobacco leaf infiltration to assess functional interactions through reporter gene activation

Comparative Analysis Strategy:

• Compare complex composition under different conditions (e.g., different sugars that induce anthocyanin accumulation)

• Assess how promoter activation correlates with complex formation

• Determine tissue-specific variations in complex composition

Research has shown that PpbHLH35 works cooperatively with PpMYB44-like to activate the PpUFGT promoter , making this specific interaction a valuable model for studying the MBW complex dynamics in anthocyanin regulation.

What are the latest techniques for multiplexed detection of BHLH35 alongside other transcription factors?

Advanced multiplexed detection techniques allow researchers to simultaneously visualize BHLH35 and interacting transcription factors:

Multiplexed Immunofluorescence Methods:

• Sequential multiplexing: Apply and strip primary antibodies sequentially

  • Use mild elution buffers (glycine pH 2.5, SDS, or heat)

  • Validate complete stripping between rounds

  • Document using different fluorophores

• Spectral unmixing: Use spectrally overlapping fluorophores with computational separation

  • Requires specialized instrumentation

  • Allows simultaneous visualization of 6-8 targets

  • Essential for co-localization studies of transcription factor complexes

• Proximity ligation assay (PLA): Detect protein-protein interactions with spatial resolution

  • Particularly valuable for studying BHLH35 interactions with MYB factors

  • Generates signal only when target proteins are within 40nm

  • Can be combined with conventional immunofluorescence

Mass Cytometry Applications:

• Metal-tagged antibodies enable simultaneous detection of 40+ proteins

• Requires specialized equipment (CyTOF)

• Particularly valuable for single-cell analysis of transcription factor networks

Imaging Mass Spectrometry:

• Combines spatial information with protein identification

• Allows label-free detection of multiple proteins

• Provides insights into tissue distribution of transcription factor complexes

These advanced techniques are particularly valuable for studying the dynamic assembly of transcription factor complexes like the MYB-bHLH-WD40 complex involved in anthocyanin regulation, where multiple protein partners interact in a tissue-specific and stimulus-dependent manner .

What are the emerging trends in BHLH35 antibody research?

The field of BHLH35 antibody research is evolving rapidly, with several emerging trends that promise to enhance our understanding of this transcription factor's role in plant development and stress responses:

Single-Cell Applications:

• Development of highly sensitive detection methods for single-cell western blotting

• Integration with single-cell transcriptomics to correlate protein levels with gene expression

• Spatial transcriptomics approaches to map BHLH35 distribution in complex tissues

Structural Biology Integration:

• Combining antibody-based purification with cryo-EM for structural studies of BHLH35 complexes

• Epitope mapping to identify critical functional domains

• Structure-guided development of more specific antibodies targeting unique regions

Systems Biology Approaches:

• Large-scale IP-MS studies to map the complete BHLH35 interactome

• Integration of ChIP-seq, RNA-seq, and proteomics data to build comprehensive regulatory networks

• Mathematical modeling of MYB-bHLH-WD40 complex dynamics in response to environmental stimuli

Therapeutic and Agricultural Applications:

• Development of antibody-based tools for manipulating anthocyanin production in crops

• Design of engineered plants with modified BHLH35 activity for enhanced stress tolerance

• Exploration of BHLH35 as a target for modulating plant secondary metabolite production

As our understanding of bHLH transcription factors continues to grow, the development of increasingly specific and versatile antibodies will remain crucial for elucidating their complex roles in plant development and environmental responses .