BHLH107 Antibody

What is bHLH107 and why is it significant in plant immunity research?

bHLH107 (Basic Helix-Loop-Helix 107) is a transcription factor in Arabidopsis thaliana that plays a critical role in copper-induced defense responses. It functions by binding to copper-responsive elements (CuREs) in the promoters of defense-related genes, particularly ACS8, which is involved in ethylene biosynthesis. The significance of bHLH107 lies in its ability to positively regulate plant immunity against bacterial pathogens such as Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) .

When plants are exposed to copper ions (Cu²⁺), bHLH107 undergoes phosphorylation at specific serine residues (Ser62 and Ser72), which prompts its translocation from the cytoplasm to the nucleus. In the nucleus, it associates with other transcription factors like HY5 to activate defense gene expression . This copper-mediated immune response represents an important mechanism by which plants can rapidly defend against pathogen attack, making bHLH107 a significant target for researchers studying plant immunity pathways.

What detection methods are available for studying bHLH107 protein localization?

Several detection methods have been validated for studying bHLH107 protein localization in plant cells:

• Cell Fractionation Assays: This technique allows researchers to separate cellular components (cytoplasmic and nuclear fractions) and detect bHLH107 protein distribution between these compartments. In published studies, this approach revealed that Cu²⁺ treatment causes bHLH107 to accumulate in the nucleus within 0.5-1 hour of exposure .

• Fluorescent Protein Fusion Systems: The construction of bHLH107-YFP fusion proteins expressed under native promoters enables visualization of the protein's subcellular localization via confocal microscopy. This approach confirmed that bHLH107-YFP predominantly localizes to the cytoplasm under normal conditions but translocates to the nucleus upon Cu²⁺ treatment .

• Immunofluorescence with Anti-bHLH107 Antibodies: While not explicitly detailed in the provided search results, antibodies against bHLH107 can be used with secondary fluorescent-labeled antibodies to visualize endogenous protein localization.

• ChIP-qPCR: Chromatin immunoprecipitation coupled with quantitative PCR using bHLH107-specific antibodies allows detection of the protein bound to DNA in the nucleus, confirming its functionality as a transcription factor. This method demonstrated that bHLH107 binds to the CuRE elements in the ACS8 promoter specifically under Cu²⁺ treatment conditions .

These methodologies provide complementary approaches to track bHLH107 movement within cells and validate its nuclear accumulation during immune responses.

How can I validate the specificity of a bHLH107 antibody for experimental use?

Validating the specificity of a bHLH107 antibody requires multiple complementary approaches:

• Western Blot Analysis with Positive and Negative Controls:

  • Use protein extracts from wild-type plants as positive controls

  • Include protein extracts from bHLH107 knockout/mutant lines (such as bhlh107-1, bhlh107-2, or CRISPR-generated bhlh107) as negative controls

  • The antibody should detect a band of the expected molecular weight (~30-35 kDa) in wild-type samples but not in knockout lines

• Immunoprecipitation Validation:

  • Perform immunoprecipitation with the bHLH107 antibody followed by mass spectrometry

  • Confirm that bHLH107 peptides are identified among the precipitated proteins

  • Check for minimal cross-reactivity with other bHLH family proteins

• Phosphorylation-State Specificity Testing:

  • If using phospho-specific antibodies for Ser62 or Ser72 of bHLH107, compare reactivity between samples with and without Cu²⁺ treatment

  • Include samples expressing phospho-mutant versions (S62/72A) and phospho-mimetic versions (S62/72D) to confirm specificity to the phosphorylation state

• Immunostaining Cross-Validation:

  • Compare immunostaining patterns using the antibody with the localization of fluorescently tagged bHLH107 (bHLH107-YFP)

  • Patterns should match, particularly regarding the cytoplasmic-to-nuclear translocation following Cu²⁺ treatment

• Pre-absorption Control:

  • Pre-incubate the antibody with purified recombinant bHLH107 protein before use in experiments

  • This should abolish specific signals if the antibody is truly specific

These validation steps ensure that experimental results obtained with the bHLH107 antibody accurately reflect the protein's behavior rather than artifacts or cross-reactivity.

What are the recommended fixation and extraction protocols for preserving bHLH107 protein integrity?

For optimal preservation of bHLH107 protein integrity during experimental procedures, consider the following protocols:

Fixation Protocol for Immunohistochemistry:

• Fix plant tissues in 4% paraformaldehyde in PBS (pH 7.4) for 20-30 minutes at room temperature

• Wash 3 times with PBS to remove excess fixative

• For phosphorylated bHLH107 detection, include phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) in all buffers to prevent dephosphorylation

• Consider adding 1 mM CaCl₂ to buffers when studying CPK3-mediated phosphorylation of bHLH107

Protein Extraction for Western Blot and Immunoprecipitation:

• Grind plant tissue in liquid nitrogen to a fine powder

• Extract proteins in buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • 10% glycerol

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) for phosphorylation studies

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

• Collect supernatant for further analysis

Subcellular Fractionation Protocol:

• Homogenize tissue in nuclear isolation buffer (NIB):

  • 10 mM MES-KOH (pH 5.5)

  • 10 mM NaCl

  • 10 mM KCl

  • 2.5 mM EDTA

  • 250 mM sucrose

  • 0.1 mM spermine

  • 0.5 mM spermidine

  • 1 mM DTT

  • Protease and phosphatase inhibitors

• Filter through nylon mesh (60 μm)

• Add Triton X-100 to 0.3% final concentration

• Incubate on ice for 15 minutes

• Centrifuge at 1,500 g for 10 minutes to pellet nuclei

• Save supernatant as cytoplasmic fraction

• Wash nuclear pellet twice with NIB + 0.3% Triton X-100

• Extract nuclear proteins with high-salt buffer (NIB + 400 mM NaCl)

These protocols are designed to maintain protein phosphorylation states and preserve protein-protein interactions that are critical for studying bHLH107 function.

How can I design experiments to detect phosphorylation-specific changes in bHLH107 using antibodies?

Designing experiments to detect phosphorylation-specific changes in bHLH107 requires sophisticated approaches combining multiple techniques:

Experimental Design for Phosphorylation Detection:

• Generation of Phospho-Specific Antibodies:

  • Develop antibodies targeting the phosphorylated Ser62 and Ser72 residues of bHLH107

  • Use synthetic phosphopeptides containing these sites as immunogens

  • Validate antibody specificity using peptide competition assays with phosphorylated and non-phosphorylated peptides

• Phos-tag SDS-PAGE Analysis:

  • Use Phos-tag acrylamide gels which specifically retard the migration of phosphorylated proteins

  • Compare migration patterns of bHLH107 from samples with and without Cu²⁺ treatment

  • Include phosphatase-treated samples as controls to confirm phosphorylation-dependent mobility shifts

  • Compare with phospho-mutant (S62/72A) and phospho-mimetic (S62/72D) versions of bHLH107

• Time-Course Experiments:

  • Treat plants with Cu²⁺ and collect samples at multiple time points (0, 15, 30, 60, 120 min)

  • Use phospho-specific antibodies to track the kinetics of Ser62 and Ser72 phosphorylation

  • Correlate phosphorylation status with nuclear localization and target gene expression

• Pharmacological Approaches:

  • Apply calcium channel blockers (LaCl₃) to inhibit CPK3 activity

  • Use kinase inhibitors to block CPK3 function

  • Assess effects on bHLH107 phosphorylation and nuclear translocation

  • Compare with cpk3 mutant plants to confirm specificity

• In Vitro Kinase Assays:

  • Express and purify recombinant CPK3 and bHLH107 (wild-type and mutant variants)

  • Perform in vitro phosphorylation reactions with [γ-³²P]ATP

  • Analyze phosphorylation by autoradiography and phospho-specific antibodies

  • Use mass spectrometry to confirm phosphorylation sites

Table 1: Recommended Experimental Controls for Phosphorylation Studies

By implementing this multi-faceted approach, researchers can comprehensively characterize the phosphorylation dynamics of bHLH107 and their functional significance in immune responses.

What are the critical considerations when performing ChIP assays with bHLH107 antibodies?

Performing Chromatin Immunoprecipitation (ChIP) assays with bHLH107 antibodies requires careful optimization and consideration of several critical factors:

Critical Considerations for bHLH107 ChIP Assays:

• Antibody Quality and Validation:

  • Ensure high specificity and affinity of anti-bHLH107 antibodies

  • Validate antibody effectiveness in immunoprecipitating bHLH107 from nuclear extracts

  • Consider using epitope-tagged bHLH107 (e.g., HA-tag) and corresponding antibodies for higher specificity

  • If studying phosphorylated forms, validate that antibodies recognize the DNA-bound phosphorylated state

• Crosslinking Optimization:

  • Formaldehyde concentration (1-1.5%) and time (10-15 minutes) must be optimized

  • Over-crosslinking can mask epitopes and reduce antibody accessibility

  • Under-crosslinking may fail to capture transient DNA-protein interactions

  • Remember that bHLH107-DNA interactions are stimulus-dependent (Cu²⁺-induced)

• Timing Considerations:

  • bHLH107 shows dynamic nuclear accumulation after Cu²⁺ treatment

  • Perform time-course experiments (0, 1, 2, 4 hours post-treatment)

  • Peak nuclear accumulation occurs at 0.5-1 hour after Cu²⁺ treatment

  • ChIP-qPCR shows enrichment of bHLH107 at the CuRE element only under Cu²⁺ treatment

• Control Regions and Background:

  • Include negative control regions without bHLH107 binding sites

  • Use mutant versions of target sequences (mutated CuRE elements)

  • Include IgG control immunoprecipitations to establish background

  • Use bhlh107 mutant plants as biological negative controls

• Co-factor Considerations:

  • bHLH107 interacts with HY5 in the nucleus

  • Consider potential cooperative binding and effects on ChIP efficiency

  • Sequential ChIP (re-ChIP) may be needed to study co-occupancy with HY5

• qPCR Primer Design:

  • Design primers flanking known CuRE elements in target promoters (e.g., ACS8)

  • Include multiple primer pairs spanning the promoter region to determine binding specificity

  • Ensure primers have similar amplification efficiency for accurate quantification

Table 2: Recommended ChIP-qPCR Controls for bHLH107 Studies

Following these considerations will help ensure reliable and reproducible ChIP results when studying bHLH107's DNA binding activity and transcriptional regulation properties.

How can I resolve contradictory data between antibody-based detection and fluorescent protein fusion localization studies of bHLH107?

Resolving contradictions between antibody-based detection and fluorescent protein fusion localization studies of bHLH107 requires systematic troubleshooting and analysis:

Methodological Approach to Resolve Contradictory Data:

• Identify Specific Inconsistencies:

  • Document precise differences in subcellular localization patterns

  • Note timing discrepancies in nuclear translocation after Cu²⁺ treatment

  • Determine if differences are qualitative (location) or quantitative (amount)

  • Assess if contradictions occur under specific experimental conditions

• Evaluate Technical Artifacts in Both Methods:

  For Antibody-Based Detection:

  • Antibody specificity: Verify using western blots with bhlh107 mutant controls

  • Fixation effects: Different fixatives may alter epitope accessibility or protein localization

  • Permeabilization issues: Insufficient permeabilization may prevent antibody access to certain compartments

  • Background signals: High background may mask subtle localization patterns

  For Fluorescent Fusion Proteins:

  • Size effects: The YFP tag (~27 kDa) may interfere with normal protein trafficking

  • Expression levels: Overexpression may saturate normal localization mechanisms

  • Functionality: Verify the fusion protein retains biological activity using complementation assays

  • Photobleaching: Weak signals may be missed due to photobleaching

• Perform Reconciliation Experiments:

  • Co-localization studies: Use anti-bHLH107 antibodies in plants expressing bHLH107-YFP

  • If signals co-localize, both methods are detecting the same protein

  • If signals differ, determine which method correlates better with functional outcomes

  • Conduct fractionation experiments followed by western blotting with both anti-bHLH107 and anti-YFP antibodies

• Evaluate Biological Variables:

  • Phosphorylation state: The S62/72 phosphorylation affects localization

  • Interaction partners: HY5 and CPK3 associations may influence localization

  • Cell type differences: Localization may vary by cell type or tissue

  • Developmental stage: Consider potential developmental regulation

• Functional Correlation Analysis:

  • Determine which localization pattern better correlates with:

    • ACS8 gene expression

    • Disease resistance phenotypes

    • Binding to CuRE elements (ChIP data)

  • The method that better correlates with functional outcomes is likely more accurate

Table 3: Systematic Comparison Framework for Resolving Localization Discrepancies

By systematically analyzing these factors, researchers can determine the source of contradictions and develop a more accurate integrated model of bHLH107 localization dynamics.

What are the most appropriate quantification methods for analyzing western blot data of phosphorylated bHLH107?

Quantifying phosphorylated bHLH107 in western blots requires specialized approaches to ensure accuracy and reliability:

Optimal Quantification Methods:

• Phos-tag Gel-Based Quantification:

  • Phos-tag SDS-PAGE separates phosphorylated from non-phosphorylated bHLH107

  • Calculate the phosphorylation ratio: Phospho-bHLH107 / Total bHLH107

  • Use densitometry software (ImageJ, Image Lab) for band intensity measurement

  • This approach allows monitoring of the phosphorylation state without phospho-specific antibodies

• Dual Antibody Approach:

  • Use phospho-specific antibodies (targeting phospho-Ser62 and phospho-Ser72)

  • Simultaneously use pan-bHLH107 antibodies on parallel blots or after stripping

  • Calculate phosphorylation ratio: Phospho-signal / Total-signal

  • Normalize using appropriate loading controls (e.g., Histone H3 for nuclear fractions, GAPDH for cytosolic fractions)

• Internal Standardization:

  • Include recombinant phosphorylated bHLH107 standards at known concentrations

  • Generate standard curves for accurate quantification

  • Use both wild-type and phospho-mimic (S62/72D) recombinant proteins as standards

  • Account for differential antibody affinities for phosphorylated vs. non-phosphorylated forms

• Statistical Analysis Considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (ANOVA with post-hoc tests for time-course studies)

  • Report variability measures (standard deviation or standard error)

  • Consider normalization issues when comparing across different experimental conditions

Table 4: Comparison of bHLH107 Phosphorylation Quantification Methods

• Advanced Considerations for Time-Course Experiments:

  • For Cu²⁺-induced phosphorylation kinetics, collect samples at precise timepoints (0, 15, 30, 60, 120 min)

  • Plot phosphorylation ratio against time to determine the rate of modification

  • Correlate phosphorylation kinetics with nuclear accumulation and target gene activation

  • Consider using curve-fitting approaches to model phosphorylation dynamics

• Validation Through Complementary Approaches:

  • Confirm western blot quantification with orthogonal methods:

    • Mass spectrometry-based phosphopeptide quantification

    • Immunoprecipitation followed by phospho-amino acid analysis

    • In vitro kinase assays with recombinant proteins

How can I study the interactions between bHLH107 and other proteins such as CPK3 and HY5 using antibody-based approaches?

Studying protein-protein interactions involving bHLH107, CPK3, and HY5 requires sophisticated antibody-based approaches:

Comprehensive Interaction Study Methods:

• Co-Immunoprecipitation (Co-IP) Strategies:

  • Standard Co-IP:

    • Immunoprecipitate bHLH107 using specific antibodies

    • Detect CPK3 or HY5 in the precipitates by western blotting

    • Perform reciprocal Co-IPs (immunoprecipitate CPK3 or HY5, detect bHLH107)

  • Stimulus-Dependent Co-IP:

    • Compare interactions before and after Cu²⁺ treatment

    • Monitor kinetics of interaction formation/dissolution

    • Include phosphorylation mutants (S62/72A, S62/72D) to assess phosphorylation dependency

  • Sequential Co-IP (Re-IP):

    • First immunoprecipitate bHLH107, then re-immunoprecipitate from the eluate using anti-CPK3 antibodies

    • This approach can identify ternary complexes (bHLH107-CPK3-HY5)

• Proximity Ligation Assay (PLA):

  • This technique enables visualization of protein interactions in situ

  • Use primary antibodies against bHLH107 and CPK3 or HY5

  • Secondary antibodies with attached oligonucleotides generate fluorescent signals only when proteins are in close proximity (<40 nm)

  • Quantify interaction signals in different subcellular compartments before and after Cu²⁺ treatment

  • This method can reveal where interactions occur within the cell (cytoplasm vs. nucleus)

• Bimolecular Fluorescence Complementation (BiFC) with Antibody Validation:

  • Express bHLH107-nYFP and CPK3-cYFP or HY5-cYFP in plant cells

  • Observe fluorescence when proteins interact

  • Use antibodies in parallel experiments to confirm the presence of both proteins

  • Compare BiFC patterns with antibody staining to validate interaction sites

• Pull-Down Assays with Recombinant Proteins:

  • Express recombinant MBP-bHLH107, GST-CPK3, and GST-HY5

  • Perform pull-down assays and detect interactions by western blotting

  • Include phosphorylation in vitro before pull-down to assess effects on interaction

  • This approach has been successfully used to demonstrate direct interactions

Table 5: Interaction Domain Mapping Strategy

• ChIP-Re-ChIP for Studying Cooperative DNA Binding:

  • First perform ChIP with anti-bHLH107 antibodies

  • Re-ChIP the eluted material with anti-HY5 antibodies

  • Analyze by qPCR targeting the ACS8 promoter

  • This approach can determine if bHLH107 and HY5 simultaneously occupy the same DNA regions

• Proteomic Analysis of Interaction Partners:

  • Perform immunoprecipitation of bHLH107 complexes from plants before/after Cu²⁺ treatment

  • Analyze by mass spectrometry to identify all interaction partners

  • Validate novel interactions using the methods described above

  • Compare interactomes between wild-type, phospho-mutant, and phospho-mimetic variants

By combining these approaches, researchers can build a comprehensive understanding of the dynamic protein interaction network involving bHLH107, CPK3, and HY5 during copper-induced immune responses.

How can bHLH107 antibodies be used to study copper-induced signaling pathways beyond plant immunity?

bHLH107 antibodies can serve as powerful tools for investigating broader copper-induced signaling pathways:

Expanded Research Applications:

• Copper Homeostasis Regulation:

  • Use bHLH107 antibodies to study potential roles in general copper sensing mechanisms

  • Investigate whether bHLH107 regulates genes involved in copper transport or detoxification

  • Compare bHLH107 activity under copper deficiency versus excess conditions

  • Examine potential crosstalk with known copper homeostasis regulators like SPL7

• Abiotic Stress Response Network Mapping:

  • Apply bHLH107 antibodies in ChIP-seq experiments across multiple stress conditions

  • Compare genome-wide binding profiles under copper treatment versus other metal stresses

  • Identify shared and unique target genes in different stress responses

  • Construct regulatory networks using bHLH107 as a node connecting different pathways

• Hormonal Crosstalk Investigation:

  • bHLH107 regulates ACS8, an ethylene biosynthesis gene

  • Use antibodies to track bHLH107 activity during treatments with multiple hormones

  • Investigate how ethylene, salicylic acid, or jasmonic acid signaling affects bHLH107 phosphorylation

  • Examine whether bHLH107 participates in hormone-mediated growth-defense tradeoffs

• Developmental Regulation Studies:

  • Track bHLH107 abundance, phosphorylation state, and nuclear localization across developmental stages

  • Investigate tissue-specific activation patterns using immunohistochemistry

  • Determine if copper-induced bHLH107 activity varies with developmental context

  • Examine potential developmental phenotypes in bHLH107 phosphorylation mutants

Table 6: Proposed Research Areas for bHLH107 Antibody Applications

• Methodological Innovations:

  • Develop proximity-dependent labeling techniques using bHLH107 antibodies

  • Create biosensor systems that report on bHLH107 activation status in real-time

  • Establish single-cell antibody-based detection methods to study cell-specific responses

  • Design high-throughput screening approaches to identify chemical modulators of bHLH107 function

By expanding research beyond the established role in immunity, bHLH107 antibodies can help unveil broader functions in copper sensing, signaling, and plant adaptation mechanisms.

What are the challenges and solutions for detecting post-translational modifications of bHLH107 beyond phosphorylation?

While phosphorylation of bHLH107 has been well-characterized, other post-translational modifications (PTMs) may also regulate its function. Here's a comprehensive analysis of challenges and solutions for studying these additional PTMs:

Challenges and Solutions for Detecting Non-Phosphorylation PTMs:

• Ubiquitination Detection:

  Challenges:

  • Ubiquitination may be transient due to rapid protein degradation

  • Multiple ubiquitination patterns (mono-, poly-, different chain linkages) add complexity

  • Antibody specificity for ubiquitinated forms is difficult to achieve

  Solutions:

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated forms

  • Perform immunoprecipitation with bHLH107 antibodies followed by ubiquitin-specific antibody detection

  • Express His-tagged ubiquitin and purify under denaturing conditions to capture all ubiquitinated forms

  • Compare ubiquitination patterns before and after Cu²⁺ treatment to identify regulation

• SUMOylation Analysis:

  Challenges:

  • Low stoichiometry of SUMOylated proteins

  • SUMO proteases may remove modifications during extraction

  • Lack of specific antibodies for SUMOylated bHLH107

  Solutions:

  • Include SUMO protease inhibitors (NEM) in extraction buffers

  • Use SUMO-specific antibodies after bHLH107 immunoprecipitation

  • Analyze bHLH107 sequence for potential SUMOylation sites using bioinformatics

  • Create SUMO-site mutants to assess functional significance

• Acetylation Characterization:

  Challenges:

  • Acetylation may be substoichiometric and site-specific

  • Antibodies against specific acetylated residues are rarely available

  • Functional significance may be context-dependent

  Solutions:

  • Use pan-acetyl-lysine antibodies after bHLH107 immunoprecipitation

  • Include deacetylase inhibitors (TSA, nicotinamide) during extraction

  • Employ mass spectrometry to map acetylation sites

  • Investigate interactions with known acetyltransferases and deacetylases

Table 7: Methodology for Detecting Multiple PTMs on bHLH107

• Strategies for Multi-PTM Analysis:

  Challenges:

  • Different PTMs may compete for the same residues

  • PTMs may show interdependence (one modification affecting another)

  • Analytical techniques often focus on single modification types

  Solutions:

  • Perform sequential immunoprecipitations with different PTM-specific antibodies

  • Use mass spectrometry approaches optimized for multiple PTM detection

  • Create a PTM "code" map relating different modifications to functional states

  • Develop computational models to predict PTM patterns under different conditions

• Redox-Based Modifications:

  Challenges:

  • Copper can induce oxidative stress, potentially causing redox-based PTMs

  • Redox modifications are often reversible and lost during sample processing

  Solutions:

  • Use alkylating agents to trap redox states during extraction

  • Compare protein mobility under reducing vs. non-reducing conditions

  • Investigate potential disulfide bond formation in response to copper

  • Study interactions with redox-regulating proteins (thioredoxins, glutaredoxins)

By systematically addressing these challenges, researchers can develop a comprehensive understanding of how multiple PTMs coordinate to regulate bHLH107 function in copper-induced signaling pathways.