BHLH130 Antibody

What is BHLH130 and why is it significant in plant research?

BHLH130 is a plant-specific transcription factor belonging to the basic helix-loop-helix (bHLH) family. It contains a conserved bHLH domain responsible for DNA binding and dimerization, with primary localization in the nucleus as confirmed by GFP fusion experiments. Its significance stems from its regulatory role in multiple stress-responsive pathways, particularly those related to drought tolerance, oxidative stress management, and nitrogen use efficiency .

MdbHLH130, an apple homolog, has been extensively characterized and shown to confer enhanced water stress tolerance through the modulation of stomatal closure and reactive oxygen species (ROS) scavenging mechanisms . The protein represents an important target for understanding molecular mechanisms of stress adaptation in crop plants.

What types of BHLH130 antibodies are available for research use?

Several companies produce polyclonal antibodies against BHLH130 and related transcription factors. Currently available antibodies include:

Custom BHLH130 antibodies can also be manufactured with specific buffer compositions (typically 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative) . Lead times for custom antibody production typically range from 14-16 weeks.

What are the optimal protocols for using BHLH130 antibodies in Western blotting experiments?

For Western blotting applications using BHLH130 antibody:

• Sample Preparation: Extract total protein from plant tissues using standard extraction buffers containing protease inhibitors.

• Protein Loading: Load 20-40 μg of total protein per lane.

• Electrophoresis and Transfer: Use standard SDS-PAGE with 10-12% gels followed by transfer to PVDF or nitrocellulose membranes.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute BHLH130 antibody in the range of 1:1,000 in blocking buffer. Incubate overnight at 4°C.

• Secondary Antibody: Use appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG at 1:5,000 to 1:10,000 dilution).

• Detection: Use enhanced chemiluminescence for visualization .

This protocol has been successfully used to validate BHLH130 overexpression in transgenic apple calli and to assess protein levels under various stress conditions .

How can BHLH130 antibodies be used in immunohistochemistry studies?

For immunohistochemistry applications:

• Tissue Fixation: Fix plant tissues in 4% paraformaldehyde or other appropriate fixative.

• Sectioning: Prepare 5-10 μm sections using a microtome.

• Antigen Retrieval: Perform heat-induced epitope retrieval if necessary.

• Blocking: Block with 3-5% BSA or appropriate blocking buffer.

• Primary Antibody: Apply BHLH130 antibody at dilutions ranging from 1:100 to 1:400, depending on the specific antibody and tissue.

• Secondary Antibody: Use fluorescently-labeled or HRP-conjugated secondary antibodies.

• Visualization: For fluorescent detection, examine using confocal microscopy; for chromogenic detection, develop using DAB substrate .

This approach has been used to confirm the nuclear localization of BHLH130 in plant tissues and to examine its expression patterns in response to various stresses .

How should BHLH130 antibodies be integrated into stress response studies?

When designing stress response experiments incorporating BHLH130 antibody analysis:

• Stress Treatment Design: Apply relevant stressors (drought, PEG6000, oxidative stress, or nitrogen limitation) in controlled conditions.

• Time-Course Sampling: Collect samples at multiple time points (e.g., 0, 1, 3, 6, 12, 24 hours) after stress application to capture dynamic changes in BHLH130 expression.

• Protein Expression Analysis: Use Western blotting with BHLH130 antibodies to quantify protein levels.

• Correlation with Physiological Parameters: Pair antibody-based protein detection with measurements of:

  • ROS-scavenging enzyme activities (SOD, POD, CAT)

  • Malondialdehyde (MDA) content (marker of oxidative damage)

  • Fresh weight changes (for growth assessment)

  • Stomatal conductance measurements

• Gene Expression Analysis: Complement protein analysis with qRT-PCR of BHLH130 and downstream target genes .

This integrated approach was successfully employed in studies showing that MdbHLH130-overexpressing apple calli exhibited 20% higher fresh weight and 35% lower MDA content compared to wild-type under 6% PEG6000 stress conditions .

How can researchers validate the specificity of BHLH130 antibodies?

To validate BHLH130 antibody specificity:

• Positive Controls: Use overexpression systems (e.g., transgenic lines overexpressing tagged BHLH130) to confirm antibody detection of the target protein.

• Negative Controls: Include knockout/knockdown lines or tissues known not to express BHLH130 to confirm absence of non-specific binding.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to samples; this should abolish specific signals.

• Molecular Weight Verification: Confirm that the detected band appears at the expected molecular weight (~30 kDa for BHLH130).

• Cross-Reactivity Assessment: Test the antibody against related bHLH family members if possible to assess cross-reactivity.

• Multiple Antibody Comparison: If available, compare results using antibodies raised against different epitopes of BHLH130 .

This comprehensive validation approach is essential given the structural similarities between different bHLH family members and potential for cross-reactivity.

What are common issues in BHLH130 antibody experiments and how can they be addressed?

Common challenges and solutions when working with BHLH130 antibodies include:

• Weak Signal:

  • Increase antibody concentration (try 1:500 instead of 1:1,000)

  • Extend primary antibody incubation time (up to 48 hours at 4°C)

  • Optimize protein extraction to preserve BHLH130 integrity

  • Consider using enhanced detection systems

• High Background:

  • Increase washing steps (5-6 washes, 10 minutes each)

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Decrease secondary antibody concentration

  • Pre-absorb antibody with plant extract from negative control samples

• Multiple Bands:

  • Increase gel resolution with longer run times

  • Test antibody on BHLH130 knockout tissue to identify non-specific bands

  • Use fresh protein samples to minimize degradation

  • Add additional protease inhibitors to extraction buffer

• Inconsistent Results:

  • Standardize protein extraction and handling procedures

  • Include internal loading controls

  • Ensure consistent stress application in experiments

  • Consider protein post-translational modifications that may affect antibody recognition

Addressing these issues requires systematic optimization and careful experimental design.

How should researchers interpret discrepancies between BHLH130 protein levels and gene expression data?

When encountering discrepancies between BHLH130 protein abundance (determined by antibody-based methods) and mRNA levels (determined by qRT-PCR):

• Time-Course Considerations: Protein synthesis typically lags behind transcriptional changes; analyze samples at multiple time points (e.g., 0, 3, 6, 12, 24, 48 hours) to capture this temporal relationship.

• Post-Transcriptional Regulation: Investigate potential microRNA regulation of BHLH130 mRNA that might affect translation efficiency.

• Protein Stability Analysis: Assess BHLH130 protein half-life using cycloheximide chase experiments with the antibody to detect degradation rates.

• Post-Translational Modifications: Consider that PTMs may affect antibody recognition or protein stability; use phospho-specific antibodies if phosphorylation is suspected.

• Cellular Compartmentalization: Use subcellular fractionation combined with antibody detection to determine if protein localization changes affect total protein measurement.

• Technical Validation: Confirm results using alternative techniques (e.g., mass spectrometry) to validate antibody-based quantification .

Studies on MdbHLH130 have shown cases where protein accumulation patterns during stress do not perfectly match transcript abundance patterns, highlighting the importance of post-transcriptional and post-translational regulatory mechanisms .

How can BHLH130 antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments?

For ChIP applications using BHLH130 antibodies:

• Crosslinking: Crosslink plant tissue with 1% formaldehyde for 10-15 minutes under vacuum.

• Chromatin Extraction and Sonication: Extract chromatin and fragment to 200-500 bp by sonication.

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with BHLH130 antibody (2-5 μg per immunoprecipitation) overnight at 4°C

  • Add protein A/G beads and incubate 2-4 hours

  • Perform stringent washing steps

• Reverse Crosslinking and DNA Purification: Reverse crosslinks and purify DNA for downstream analysis.

• Target Validation: Perform qPCR with primers specific to promoter regions of known or putative BHLH130 target genes (e.g., ROS-scavenging genes like SOD, POD, and CAT).

• Controls: Include input chromatin and non-specific IgG controls.

• Genome-Wide Analysis: For comprehensive target identification, combine with sequencing (ChIP-seq) .

This approach would be valuable for identifying direct transcriptional targets of BHLH130, particularly genes involved in stress response pathways like ROS-scavenging systems and flavonoid biosynthesis.

What strategies enable researchers to study BHLH130 protein-protein interactions?

To investigate BHLH130 protein-protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Extract proteins under native conditions

  • Incubate protein extract with BHLH130 antibody (2-5 μg)

  • Precipitate using protein A/G beads

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

  • Include appropriate controls (non-specific IgG, input sample)

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of BHLH130 and candidate interacting proteins with split fluorescent protein fragments

  • Co-express in plant systems (e.g., Nicotiana benthamiana leaves)

  • Visualize interaction-dependent fluorescence

  • Validate using BHLH130 antibodies in parallel experiments

• Proximity-Dependent Biotin Identification (BioID):

  • Create fusion proteins of BHLH130 with a biotin ligase

  • Express in plant tissues

  • Identify biotinylated proteins (proximal to BHLH130) using streptavidin pulldown

  • Confirm results using BHLH130 antibodies in conventional co-IP experiments

• Yeast Two-Hybrid Validation:

  • Screen for interactors using Y2H

  • Validate interactions in planta using BHLH130 antibodies in co-IP experiments

Research has indicated BHLH130's interaction with ERF109, a key regulator of flavonoid pathways, which could be further validated and characterized using these approaches.

How can researchers leverage BHLH130 antibodies in multi-omics integration studies?

For integrating BHLH130 antibody-based analyses with multi-omics approaches:

• Proteomics Integration:

  • Use BHLH130 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare BHLH130 protein levels (by Western blot) with global proteome changes

  • Identify post-translational modifications of BHLH130 using modification-specific antibodies

  • Correlate with changes in interacting partners

• Transcriptomics Correlation:

  • Correlate BHLH130 protein levels with RNA-seq data

  • Perform ChIP-seq using BHLH130 antibodies to identify direct targets

  • Compare binding sites with differential expression patterns

  • Identify regulatory networks centered on BHLH130

• Metabolomics Analysis:

  • Correlate BHLH130 levels with changes in metabolite profiles

  • Focus on flavonoid, lignin, and antioxidant pathway metabolites

  • Link specific metabolic changes to BHLH130-regulated genes

• Physiological Parameters:

  • Correlate BHLH130 protein dynamics with physiological measurements

  • Track relationships between protein levels and stress tolerance metrics

  • Develop predictive models of stress response based on BHLH130 expression

This integrative approach was successfully applied in a study examining MdbHLH130's role in enhancing flavonoid biosynthesis while suppressing lignin production under low-nitrogen stress, improving nitrogen absorption in apple rootstocks .

What considerations are important when designing antibody-based assays to study BHLH130 in different plant species?

When adapting BHLH130 antibody-based assays across plant species:

• Epitope Conservation Analysis:

  • Perform sequence alignment of BHLH130 homologs across target species

  • Focus on conservation within the epitope region recognized by the antibody

  • Consider generating species-specific antibodies if conservation is low

• Validation in Each Species:

  • Test antibody specificity in each plant species before conducting experiments

  • Use transgenic lines (overexpression and knockdown) as controls where available

  • Perform Western blotting to confirm detection at the appropriate molecular weight

• Protocol Optimization:

  • Adjust extraction buffers based on species-specific tissue composition

  • Optimize antibody dilutions for each species (typically starting with 1:500-1:1,000)

  • Modify incubation times and washing conditions as needed

• Crossreactivity Assessment:

  • Test for crossreactivity with other bHLH family members in the target species

  • Perform immunoprecipitation followed by mass spectrometry to confirm specificity

  • Consider competitive binding assays with recombinant BHLH130 proteins

This cross-species approach is particularly valuable as BHLH130 functions have been demonstrated in diverse plants including apple (Malus domestica) and tobacco (Nicotiana), suggesting conserved roles in stress response across plant families .

How might BHLH130 antibodies contribute to understanding stress response signaling cascades?

BHLH130 antibodies can advance our understanding of stress signaling through:

• Temporal Dynamics Analysis:

  • Track BHLH130 protein levels at high temporal resolution during stress onset and recovery

  • Correlate with activation of upstream kinases and downstream transcriptional targets

  • Identify critical time points for intervention to enhance stress tolerance

• Subcellular Trafficking Studies:

  • Use immunofluorescence with BHLH130 antibodies to track protein movement

  • Monitor nuclear-cytoplasmic shuttling under different stress conditions

  • Identify regulatory mechanisms controlling BHLH130 localization

• Post-Translational Modification Mapping:

  • Develop modification-specific antibodies (phospho-BHLH130, etc.)

  • Determine how modifications affect BHLH130 activity and target gene selection

  • Link specific modifications to upstream stress perception events

• Stress-Specific Interactome Analysis:

  • Compare BHLH130 protein complexes under different stresses (drought, oxidative, nitrogen)

  • Identify stress-specific co-factors that modify BHLH130 function

  • Map signaling networks converging on BHLH130

Research has shown that MdbHLH130 integrates multiple stress-responsive pathways, regulating both ROS-scavenging systems and stomatal regulation, suggesting its role as a hub in stress response networks .

What methodological advances might improve BHLH130 antibody specificity and sensitivity?

Emerging methodologies to enhance BHLH130 antibody performance include:

• Single-Domain Antibody Development:

  • Generate camelid-derived single-domain antibodies (nanobodies) against BHLH130

  • Enhance penetration into plant tissues and subcellular compartments

  • Improve stability under varying experimental conditions

• Epitope-Specific Antibody Engineering:

  • Design antibodies targeting unique regions of BHLH130 to reduce cross-reactivity

  • Apply biophysics-informed models to predict and enhance antibody specificity

  • Validate using phage display technologies to screen for optimal binding properties

• Proximity-Based Detection Systems:

  • Develop split reporter systems fused to anti-BHLH130 antibody fragments

  • Enable real-time monitoring of BHLH130 in living plant tissues

  • Enhance signal-to-noise ratio through proximity-induced reporter activation

• High-Throughput Screening Platforms:

  • Develop antibody arrays for simultaneous detection of BHLH130 and interacting partners

  • Apply machine learning approaches to optimize antibody binding characteristics

  • Implement automated validation pipelines to assess specificity across conditions

These methodological advances could significantly enhance our ability to study BHLH130 dynamics in complex plant systems under varying stress conditions.