BHLH95 Antibody

What is SlbHLH95 and why would researchers use antibodies against it?

SlbHLH95 is a basic helix-loop-helix transcription factor that functions as a negative regulator of trichome formation in tomato plants (Solanum lycopersicum). Research has established that SlbHLH95 represses gibberellin (GA) biosynthesis by directly binding to the promoters of GA biosynthesis genes, particularly SlGA20ox2 and SlKS5 . Researchers would use antibodies against SlbHLH95 to:

• Detect and quantify SlbHLH95 protein expression in various tissues

• Determine its subcellular localization through immunofluorescence

• Analyze protein-protein interactions through co-immunoprecipitation

• Investigate chromatin binding patterns using ChIP assays

The development of specific antibodies against SlbHLH95 would be particularly valuable for investigating its regulatory role in plant development, especially in relation to trichome formation and gibberellin signaling pathways .

How should I design control experiments when using BHLH95 antibodies?

When using BHLH95 antibodies, robust control experiments are essential to ensure specificity and validity of results:

Recommended Controls:

For optimal experimental design, incorporate both genetic approaches (using knockout/knockdown lines) and orthogonal approaches that rely on known information about SlbHLH95 . Studies have shown that genetic validation strategies are particularly robust, with approximately 80% of antibodies validated using genetic strategies performing as expected in subsequent testing .

How can I validate a new BHLH95 antibody using genetic approaches?

Validating a BHLH95 antibody through genetic approaches requires careful experimental design following established protocols:

Step-by-Step Validation Protocol:

• Generate Knockout Controls:

  • Create SlbHLH95 knockout lines using CRISPR-Cas9 technology in appropriate tomato cell lines

  • Verify knockout at genomic level through sequencing

  • Confirm absence of SlbHLH95 transcripts through RT-qPCR

• Parallel Testing Strategy:

  • Test antibody on both wild-type and knockout samples simultaneously

  • Run Western blot analysis using standardized protein quantities

  • For immunofluorescence, use a mosaic approach with wild-type and knockout cells in the same visual field to minimize imaging biases

• Multi-Application Validation:

  • Test antibody performance in Western blot, immunoprecipitation, and immunofluorescence

  • Note that research indicates success in immunofluorescence correlates strongly with performance in other applications

• Documentation and Reporting:

  • Document all validation data methodically

  • Include complete methodology, antibody concentration, incubation conditions, and detection methods

  • Share validation data openly through repositories like ZENODO

Research indicates that for transcription factors like BHLH95, genetic validation approaches significantly outperform orthogonal approaches, especially for immunofluorescence applications where only 38% of antibodies validated by orthogonal methods were confirmed when tested against knockout controls .

What methodological approaches should I use to determine if a BHLH95 antibody can detect protein-DNA interactions?

To investigate BHLH95 protein-DNA interactions using antibodies, consider these methodological approaches:

Chromatin Immunoprecipitation (ChIP) Optimization:

• Preliminary Validation:

  • First confirm antibody specificity in Western blot and standard immunoprecipitation

  • Verify recognition of native (non-denatured) BHLH95 protein

• ChIP Protocol Development:

  • Based on BHLH95's established binding to E-box (CANNTG) motifs in promoters

  • Design primers spanning the E-box regions of target genes (e.g., SlGA20ox2, SlKS5)

  • Include control primers for non-target regions

• Sequential Validation:

  • Perform ChIP using overexpression and knockout models

  • Include spike-in controls to normalize across samples

  • Verify enrichment at known binding sites (E-box motifs) versus random genomic regions

• Complementary Approaches:

  • Supplement ChIP data with in vitro binding assays (e.g., EMSA)

  • Confirm findings with transactivation assays as performed in previous BHLH95 studies

  • Consider ChIP-seq for genome-wide binding profile

Research on BHLH95 has demonstrated its direct binding to promoters of GA biosynthesis genes via E-box motifs, making these regions primary targets for ChIP validation . Complementary approaches like yeast one-hybrid and transactivation assays can provide additional validation of protein-DNA interactions observed in ChIP experiments.

How should experimental conditions be optimized for BHLH95 antibody applications in Western blot analysis?

Optimizing Western blot conditions for BHLH95 antibody requires systematic testing of multiple parameters:

Optimization Protocol:

• Sample Preparation:

  • For intracellular proteins like BHLH95, use appropriate lysis buffers with protease inhibitors

  • Include both nuclear extraction and whole cell lysates for comparison

  • Test both denaturing and non-denaturing conditions to determine optimal epitope presentation

• Blocking and Antibody Conditions:

  • Test multiple blocking agents (BSA, milk, commercial blockers)

  • Perform antibody titration series (typically 1:100 to 1:5000)

  • Test various incubation times (2 hours at room temperature versus overnight at 4°C)

• Detection Optimization:

  • Compare chemiluminescent, fluorescent, and colorimetric detection methods

  • When using multiple antibodies, consider sequential detection strategies

  • Optimize exposure times to ensure signal is in linear range

• Controls and Validation:

  • Include positive control (tissue with high BHLH95 expression)

  • Include negative control (BHLH95 knockout lines)

  • Test antibody lot-to-lot variability to ensure reproducibility

Since BHLH95 is a transcription factor that localizes to the nucleus , particular attention should be paid to nuclear extraction protocols. Additionally, the predicted molecular weight should be verified, and any post-translational modifications that might affect mobility should be considered.

What are the recommended approaches for immunofluorescence detection of BHLH95 in plant tissues?

Immunofluorescence detection of BHLH95 in plant tissues requires specialized protocols:

Plant Tissue Immunofluorescence Protocol:

• Tissue Preparation:

  • Fixation: Use 4% paraformaldehyde with optimal penetration time for plant tissues

  • Embedding and Sectioning: Paraffin embedding for standard histology or cryo-sectioning for sensitive epitopes

  • Antigen Retrieval: Test multiple methods (heat-induced, enzymatic) to optimize epitope accessibility

• Background Reduction Strategies:

  • Autofluorescence Control: Include unstained sections to document natural plant autofluorescence

  • Blocking: Extended blocking (2+ hours) with sera matched to secondary antibody species

  • Additional Blocking: Add 0.1-0.3% Triton X-100 for membrane permeabilization

• Antibody Application:

  • Primary Antibody: Test concentration range (1:50 to 1:500)

  • Incubation: Extended incubation (overnight at 4°C) to improve penetration

  • Washing: Multiple extended washes (30 minutes each) to reduce background

• Validation Approaches:

  • Specificity Control: Compare wild-type and BHLH95 knockout tissues

  • Localization Verification: Co-label with nuclear markers (DAPI, histone antibodies)

  • Negative Controls: Secondary antibody only; isotype control; peptide competition

Previous research has shown that BHLH95 localizes to the nucleus , so nuclear staining should be prominent. Imaging should include high-resolution confocal microscopy to accurately determine subcellular localization and co-localization with other nuclear proteins.

How can I differentiate between specific and non-specific signals when using BHLH95 antibodies?

Differentiating between specific and non-specific signals requires systematic analytical approaches:

Signal Specificity Analysis Framework:

• Molecular Weight Verification:

  • BHLH95 has a predicted molecular weight that should be consistent across experiments

  • Multiple bands may indicate splice variants, post-translational modifications, or non-specific binding

  • Create a molecular weight map of expected versus observed bands

• Comparative Analysis:

  • Pattern Consistency: Compare signal patterns across different tissues with known BHLH95 expression profiles

  • Genetic Models: Compare wild-type versus knockout samples

  • Tissue Correlation: Match antibody signals with known mRNA expression patterns

• Competition and Blocking Controls:

  • Peptide Competition: Pre-incubation with immunizing peptide should abolish specific signals

  • Absorption Controls: Pre-absorption with recombinant BHLH95 protein

  • Cross-Blocking: Test if different antibodies against BHLH95 block each other's binding

• Quantitative Assessment:

  • Signal-to-Noise Ratio: Calculate across different antibody concentrations

  • Dilution Linearity: Specific signals should decrease proportionally with sample dilution

  • Replicate Consistency: Establish technical and biological variability thresholds

Since BHLH95 expression is tissue-specific with lowest expression in stems where trichomes are present at high density , comparing antibody signal patterns with this known expression profile provides a valuable specificity indicator.

What approaches should I use to analyze BHLH95 protein levels in relation to trichome development?

Analyzing BHLH95 protein levels in relation to trichome development requires integrated experimental approaches:

Multi-level Analysis Protocol:

• Developmental Time Course Analysis:

  • Collect tissue samples at defined developmental stages of trichome formation

  • Quantify BHLH95 protein levels using calibrated Western blot analysis

  • Correlate protein levels with trichome density measurements across developmental stages

• Tissue-Specific Protein Extraction:

  • Use laser capture microdissection to isolate trichome-specific tissues

  • Compare BHLH95 levels between trichome-forming and non-trichome tissues

  • Normalize protein levels to appropriate housekeeping proteins

• Correlation Analysis:

  • Analyze relationship between BHLH95 protein levels and:

    • Trichome density (types I and V)

    • GA biosynthesis gene expression (SlGA20ox2, SlKS5)

    • Trichome-related gene expression (SlCycB2, SlGAMYB2, SlGASA4, SlANT1)

• Functional Manipulation:

  • Compare protein levels across wild-type, BHLH95 overexpression, and BHLH95 knockout lines

  • Assess changes following GA treatment, which rescues the glabrous phenotype

  • Create a quantitative model of BHLH95-GA-trichome relationships

Previous research has established that BHLH95 acts as a negative regulator of trichome formation, with overexpression resulting in dramatically reduced trichome density . Statistical analysis should include regression models to quantify the relationship between BHLH95 protein levels and trichome formation metrics.

How can contradictory results from different BHLH95 antibodies be reconciled and interpreted?

When faced with contradictory results from different BHLH95 antibodies, apply this systematic reconciliation framework:

Antibody Reconciliation Approach:

• Epitope Mapping Analysis:

  • Determine the specific epitopes recognized by each antibody

  • Assess potential for epitope masking in different experimental conditions

  • Consider epitope accessibility in different protein conformations or complexes

• Methodological Cross-Validation:

  • Test all antibodies under identical conditions in parallel

  • Apply multiple detection techniques (Western blot, IP, IF) with each antibody

  • Use orthogonal approaches to verify key findings (e.g., mass spectrometry)

• Systematic Variable Testing:

  • Create a matrix of conditions (fixation methods, buffers, detergents, etc.)

  • Test each antibody across all condition combinations

  • Identify condition-dependent performance patterns

• Integrated Data Analysis:

  • Weight results based on antibody validation quality

  • Develop consensus models that incorporate all data points

  • Use Bayesian approaches to estimate probability of different interpretations

Research has shown that approximately 20-30% of protein studies use ineffective antibodies , highlighting the importance of comprehensive validation. Studies comparing antibody performance have demonstrated that genetic validation approaches (using knockout controls) provide the most reliable assessment of antibody specificity and performance .

What strategies should be employed to develop a highly specific monoclonal antibody against BHLH95?

Developing a highly specific monoclonal antibody against BHLH95 requires strategic antigen design and rigorous screening:

Strategic Development Protocol:

• Antigen Design Considerations:

  • Target unique regions of BHLH95 not conserved in other bHLH family members

  • Avoid regions that participate in DNA binding (HLH domain) that may be conserved

  • Consider developing antibodies against multiple epitopes:

    • N-terminal region (often less conserved)

    • C-terminal region (typically involved in protein-specific functions)

    • Unique internal sequences identified through alignment analysis

• Production Strategy:

  • Express recombinant protein fragments representing distinct domains

  • Synthesize unique peptides conjugated to carrier proteins

  • Consider native conformation preservation for tertiary structure epitopes

• Hybridoma Screening Process:

  • Primary Screen: ELISA against immunizing antigen

  • Secondary Screen: Western blot against wild-type versus BHLH95 knockout lysates

  • Tertiary Screen: Application-specific testing (IP, IF, ChIP)

  • Cross-reactivity Elimination: Screen against related bHLH family members

• Validation and Characterization:

  • Epitope Mapping: Determine precise binding regions

  • Affinity Measurement: Quantify binding kinetics via SPR or BLI

  • Specificity Assessment: Test against tissue panels with variable BHLH95 expression

  • Functional Testing: Verify ability to detect BHLH95-DNA interactions