BHLH149 Antibody

What is BHLH149 and why are antibodies against it important?

BHLH149 is a basic helix-loop-helix transcription factor (Uniprot No. O80482) found in Arabidopsis thaliana. This protein belongs to a family of transcription factors that regulate various developmental processes and stress responses in plants. Antibodies against BHLH149 are important research tools because they allow scientists to:

• Track the spatial and temporal expression patterns of the protein across different tissues and developmental stages

• Investigate protein-protein interactions involving BHLH149

• Assess post-translational modifications that might regulate BHLH149 activity

• Validate genetic experiments through protein detection methods

The availability of specific antibodies against plant proteins like BHLH149 is critical for advancing our understanding of plant molecular biology, particularly as we move toward more integrative systems biology approaches that require knowledge of protein localization and interaction networks .

What applications are BHLH149 antibodies validated for?

BHLH149 antibodies have been specifically validated for enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) applications. These validation processes ensure that the antibody reliably detects the target protein in these specific experimental contexts . The applications include:

• Western Blotting (WB): For detecting BHLH149 protein in plant tissue extracts and determining its molecular weight, expression levels, and potential modifications.

• ELISA: For quantitative analysis of BHLH149 protein levels in solution.

It's important to note that these antibodies have not been validated for other common applications such as immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), immunohistochemistry (IHC), or immunofluorescence (IF). Researchers wishing to use the antibody for these applications would need to perform their own validation experiments .

How should BHLH149 antibodies be stored and handled?

Proper storage and handling of BHLH149 antibodies are crucial for maintaining their activity and specificity. Based on manufacturer recommendations, the following protocols should be followed:

• Storage Temperature: Upon receipt, store at -20°C or -80°C to preserve antibody activity and prevent degradation .

• Avoid Freeze-Thaw Cycles: Repeated freezing and thawing can denature antibodies and reduce their effectiveness, so aliquoting before storage is recommended.

• Storage Buffer: The antibodies are supplied in a liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 .

• Working Dilutions: Optimal dilutions must be determined experimentally but typically range from 1:500 to 1:2000 for Western blotting applications.

For long-term storage, maintaining a stable temperature is critical. Any antibody solution removed for immediate use should be kept on ice during the experimental procedure to minimize degradation.

What are the key specifications of commercially available BHLH149 antibodies?

Commercial BHLH149 antibodies have specific characteristics that researchers should consider when selecting them for experiments:

This polyclonal antibody was generated by immunizing rabbits with recombinant BHLH149 protein and subsequently purified using antigen affinity methods, which enhances its specificity for the target protein .

How can I optimize Western blotting protocols specifically for BHLH149 detection?

Optimizing Western blotting protocols for BHLH149 detection requires careful consideration of several experimental parameters:

Sample Preparation:

• Extract proteins from Arabidopsis tissues using a buffer containing protease inhibitors to prevent degradation

• Consider using phosphatase inhibitors if investigating phosphorylation status

• Optimize protein loading (typically 20-50 μg total protein per lane)

Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution of BHLH149 (molecular weight should be verified)

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for plant proteins)

• Use wet transfer for higher molecular weight proteins or semi-dry for faster protocols

Antibody Incubation:

• Begin with a 1:1000 dilution in 5% non-fat dry milk or BSA in TBST

• Perform titration experiments (1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration

• Incubate with primary antibody overnight at 4°C for best results

• Use an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

Detection Optimization:

• Use enhanced chemiluminescence (ECL) detection systems

• Consider longer exposure times if signal is weak

• For quantitative analysis, ensure the signal is within the linear range

This optimization approach has been shown to significantly improve detection rates in plant antibody research, similar to the methods reported for other Arabidopsis proteins .

What controls should be included when working with BHLH149 antibodies?

Rigorous experimental design requires appropriate controls when working with BHLH149 antibodies:

Positive Controls:

• Recombinant BHLH149 protein (if available)

• Extracts from tissues known to express BHLH149 (based on transcriptomic data)

• Overexpression lines of BHLH149 in Arabidopsis or heterologous systems

Negative Controls:

• Extracts from bhlh149 knockout/knockdown mutant plants

• Pre-immune serum control (at the same dilution as the primary antibody)

• Secondary antibody-only control (omitting primary antibody)

• Blocking peptide competition assay (pre-incubating antibody with immunizing peptide)

Additional Validation Controls:

• Testing antibody specificity against related BHLH family members

• Cross-reactivity assessment with proteins from non-target species

• Validation across multiple experimental replicates and biological samples

Implementation of these controls follows best practices demonstrated in plant antibody research, where affinity purification and thorough validation significantly improved detection confidence rates from less than 20% to over 55% .

How can BHLH149 antibodies be used for protein localization studies?

Although BHLH149 antibodies have not been specifically validated for immunolocalization techniques, they could potentially be adapted for these applications with appropriate optimization and validation:

Immunocytochemistry Optimization Strategy:

• Fixation protocol development:

  • Test different fixatives (4% paraformaldehyde, glutaraldehyde combinations)

  • Optimize fixation times (15 min to 2 hours)

  • Evaluate various embedding mediums for plant tissues

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Enzymatic antigen retrieval (proteinase K treatment)

  • Testing different concentrations and incubation times

• Signal amplification:

  • Tyramide signal amplification

  • Avidin-biotin complex (ABC) method

  • Fluorophore-conjugated secondary antibodies of varying brightness

• Validation approaches:

  • Co-localization with known nuclear markers (for transcription factors)

  • Comparison with fluorescent protein fusion localization patterns

  • Absence of signal in knockout mutants

This methodological approach is based on successful strategies used for other plant proteins, where approximately 31% of generated antibodies were successfully developed to immunocytochemistry grade through similar optimization steps .

What are the challenges in raising antibodies against plant transcription factors like BHLH149?

Developing effective antibodies against plant transcription factors presents several unique challenges:

Technical Challenges:

• Low abundance: Transcription factors typically exist in low quantities within cells, making detection difficult

• Conservation: High sequence similarity between related BHLH family members can lead to cross-reactivity issues

• Conformational epitopes: Native protein folding may present different epitopes than those recognized in denatured WB conditions

• Post-translational modifications: PTMs may mask epitopes or create new ones in vivo

Strategic Solutions:

• Immunogen design optimization:

  • Use unique peptide sequences specific to BHLH149

  • Develop recombinant proteins with correctly folded domains

  • Consider multiple immunization strategies in parallel

• Purification approaches:

  • Implement rigorous affinity purification protocols

  • Use negative selection against related family members

  • Perform epitope mapping to characterize antibody binding sites

• Validation thoroughness:

  • Test against multiple plant tissues and developmental stages

  • Validate across different experimental techniques

  • Perform cross-reactivity tests with related BHLH proteins

This challenging landscape is reflected in plant antibody development success rates, where comprehensive studies have shown that only about 55% of antibodies raised against plant proteins successfully detect their targets with high confidence, and even fewer (approximately 31%) reach immunocytochemistry-grade quality .

What are common issues in Western blotting with BHLH149 antibodies and how can they be resolved?

When performing Western blotting with BHLH149 antibodies, researchers may encounter several common issues that can be systematically addressed:

No Signal or Weak Signal:

• Cause: Insufficient antibody concentration, protein degradation, inefficient transfer

• Solution: Increase antibody concentration, add fresh protease inhibitors, optimize transfer conditions

• Approach: Implement a gradient of antibody dilutions (1:250 to 1:2000) to determine optimal concentration

Multiple Bands:

• Cause: Cross-reactivity with related BHLH proteins, protein degradation, non-specific binding

• Solution: Increase blocking concentration, optimize washing steps, validate with knockout controls

• Technique: Pre-absorb antibody with Arabidopsis total protein extract from bhlh149 mutant before use

High Background:

• Cause: Insufficient blocking, excessive antibody concentration, inadequate washing

• Solution: Extend blocking time, decrease antibody concentration, increase washing duration

• Method: Test alternative blocking agents (5% BSA vs. non-fat milk) and TBST washing buffer with varying Tween-20 concentrations

Inconsistent Results:

• Cause: Batch-to-batch variation in antibody preparation, inconsistent sample preparation

• Solution: Use the same antibody lot for comparative experiments, standardize protein extraction protocols

• Practice: Implement internal loading controls and normalize BHLH149 signal to total protein or housekeeping proteins

These troubleshooting approaches are consistent with best practices in plant antibody research, where optimization of experimental conditions significantly improves detection confidence .

How can I evaluate and validate the specificity of BHLH149 antibodies?

Thorough validation of BHLH149 antibody specificity is essential for generating reliable research results:

Genetic Validation Approaches:

• Knockout/knockdown comparison:

  • Compare signal between wild-type and bhlh149 mutant plant extracts

  • Signal should be absent or significantly reduced in mutant samples

  • Include heterozygous plants to demonstrate dose-response relationship

• Overexpression validation:

  • Test antibody against samples from BHLH149 overexpression lines

  • Signal intensity should correlate with expression level

  • Compare native vs. tagged protein detection patterns

Biochemical Validation Methods:

• Peptide competition assay:

  • Pre-incubate antibody with excess of immunizing peptide/protein

  • Should eliminate or significantly reduce specific signal

  • Use unrelated peptide as negative control

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm that pulled-down protein corresponds to BHLH149

  • Identify potential cross-reacting proteins

Cross-Reactivity Assessment:

• Testing against related proteins:

  • Express and purify related BHLH family members

  • Test antibody reactivity against protein panel

  • Quantify relative binding affinity

This comprehensive validation strategy follows approaches that have demonstrated significant improvements in antibody specificity determination, particularly for plant research where traditional validation methods may be more challenging .

How do experimental conditions affect the performance of BHLH149 antibodies?

Various experimental parameters can significantly impact the performance of BHLH149 antibodies:

Buffer Composition Effects:

Temperature Considerations:

• Primary antibody incubation at 4°C overnight typically yields better signal-to-noise ratio

• Room temperature incubations (1-2 hours) may be sufficient but increase background

• Higher temperatures (37°C) are generally not recommended for plant antibodies

Incubation Time Impact:

• Extended primary antibody incubation (16-24 hours) often improves sensitivity

• Secondary antibody incubation is optimal at 1-2 hours at room temperature

• Washing steps should be at least 3 × 5 minutes with gentle agitation

Sample Preparation Factors:

• Fresh tissue extraction generally yields better results than frozen material

• Denaturing conditions (SDS, heat) may expose epitopes that are hidden in native conditions

• Different extraction buffers may solubilize BHLH149 with varying efficiency

These experimental considerations align with observed performance variations in plant antibody studies, where methodological optimization significantly impacts detection success rates .

What alternatives exist when BHLH149 antibodies do not perform as expected?

When BHLH149 antibodies fail to deliver expected results, researchers have several alternative approaches:

Alternative Detection Strategies:

• Epitope tagging approaches:

  • Generate transgenic plants expressing tagged BHLH149 (HA, FLAG, MYC tags)

  • Use commercially validated tag antibodies for detection

  • Consider the impact of tags on protein function and localization

• Fluorescent protein fusions:

  • Create BHLH149-GFP/RFP fusion constructs under native promoter

  • Use for in vivo localization and dynamics studies

  • Validate functionality of fusion proteins by complementation tests

Indirect Detection Methods:

• RNA-based approaches:

  • RT-qPCR for transcript level analysis as proxy for protein expression

  • RNA in-situ hybridization for tissue-specific expression patterns

  • RNA-seq for global expression profiling

• Interactome studies:

  • Yeast two-hybrid screening to identify interaction partners

  • Tandem affinity purification followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

Alternative Antibody Options:

• Custom antibody development:

  • Design multiple immunogens targeting different regions of BHLH149

  • Consider different host species to overcome potential immunogenic constraints

  • Implement rigorous affinity purification strategies

• Cross-reactive antibody utilization:

  • Test antibodies against closely related BHLH proteins

  • Validate specificity in your experimental system

  • Use in combination with genetic approaches for confirmation

These alternative approaches reflect the need for adaptability in plant molecular biology research, where success rates with antibody-based detection can be variable and may require complementary methodologies .

How can BHLH149 antibodies be integrated into multi-omics research approaches?

BHLH149 antibodies can serve as valuable tools within integrated multi-omics research frameworks:

Proteomics Integration:

• Use for validation of mass spectrometry-identified BHLH149 peptides

• Employ in targeted proteomics approaches (SRM/MRM) for quantification

• Apply in immunoprecipitation followed by mass spectrometry to identify protein complexes

• Correlate protein abundance with transcript levels from transcriptomics

Functional Genomics Applications:

• Validate knockout/knockdown efficiency at protein level

• Assess protein abundance changes in various mutant backgrounds

• Correlate phenotypic alterations with protein expression patterns

• Monitor protein expression in response to environmental stimuli

Systems Biology Framework:

• Map BHLH149 protein localization data to interaction networks

• Integrate protein expression data with metabolomic profiles

• Use for validation of computational predictions of protein function

• Apply in time-series experiments to capture dynamic protein behavior

This integrated approach to utilizing antibodies aligns with current systems biology frameworks that aim to model multi-cellular systems using comprehensive data integration, where protein localization and interaction data are crucial components .

What emerging techniques could enhance the utility of BHLH149 antibodies in plant research?

Several cutting-edge techniques could expand the applications of BHLH149 antibodies in plant molecular biology:

Advanced Microscopy Approaches:

• Super-resolution microscopy:

  • STORM/PALM techniques for nanoscale localization

  • Structured illumination microscopy (SIM) for improved resolution

  • Expansion microscopy for physical sample enlargement

• Live cell imaging optimization:

  • Antibody fragment (Fab) labeling of proteins in living cells

  • Nanobody development against BHLH149 for reduced size and improved penetration

  • SNAP/CLIP-tag systems for pulse-chase protein dynamics

Novel Biochemical Methods:

• Proximity-dependent methods:

  • Antibody-based BioID/TurboID for identifying neighboring proteins

  • APEX-based proximity labeling for ultrastructural localization

  • Split antibody complementation for protein-protein interaction studies

• Single-cell approaches:

  • Antibody-based single-cell proteomics

  • Mass cytometry (CyTOF) adaptation for plant cells

  • Spatial transcriptomics combined with protein detection

Computational Enhancement:

• AI-assisted image analysis:

  • Machine learning algorithms for automated protein localization

  • Pattern recognition for subtle expression changes

  • Quantitative image analysis across large datasets

• Integrative modeling:

  • Prediction of antibody binding sites through structural modeling

  • Epitope prediction algorithms for improved antibody design

  • In silico screening of potential cross-reactivity

These emerging techniques represent the frontier of plant molecular biology research, where improved tools for protein detection and localization are critical for advancing our understanding of complex cellular systems .

How can researchers distinguish between different isoforms or modified forms of BHLH149?

Distinguishing between BHLH149 isoforms or post-translationally modified variants requires specialized approaches:

Isoform Differentiation Strategies:

• Isoform-specific antibody development:

  • Design peptides spanning unique exon junctions

  • Target isoform-specific regions for immunization

  • Validate using overexpression of specific isoforms

• Electrophoretic resolution:

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated forms

  • Employ high-percentage gels for separating small size differences

  • Implement 2D gel electrophoresis (isoelectric focusing + SDS-PAGE)

Post-translational Modification Detection:

• Modification-specific antibodies:

  • Phospho-specific antibodies if phosphorylation sites are known

  • Ubiquitin/SUMO modification detection using co-immunoprecipitation

  • Glycosylation detection using lectins combined with antibody detection

• Enzymatic treatments:

  • Phosphatase treatment to collapse phosphorylated forms

  • Deglycosylation enzymes to remove sugar modifications

  • Comparison of mobility shifts before and after treatment

Combined Analytical Approaches:

• Mass spectrometry integration:

  • Immunoprecipitation followed by targeted MS

  • Parallel reaction monitoring for specific modification sites

  • SILAC labeling for quantitative comparisons

• Cellular fractionation:

  • Nuclear vs. cytoplasmic extraction to identify location-specific forms

  • Detergent solubility fractionation for membrane-associated variants

  • Chromatin fractionation for DNA-bound transcription factor

These methodological approaches build upon established techniques in protein biochemistry adapted to the challenges of plant proteins, where post-translational modifications often play critical roles in regulating transcription factor activity .

What considerations are important when designing experiments to study BHLH149 protein-protein interactions?

Investigating BHLH149 protein-protein interactions requires careful experimental design and consideration of several factors:

Antibody-Based Interaction Studies:

• Co-immunoprecipitation optimization:

  • Test different lysis buffers to preserve protein complexes

  • Optimize antibody concentration and incubation conditions

  • Consider crosslinking to stabilize transient interactions

  • Include appropriate negative controls (IgG, knockout extracts)

• Proximity ligation assay (PLA) adaptation:

  • Requires antibodies raised in different species

  • Optimize fixation to preserve protein complexes

  • Validate antibody specificity in the PLA context

  • Include appropriate controls for signal specificity

Complementary Approaches:

• Yeast-based methods:

  • Yeast two-hybrid screening or validation

  • Split-ubiquitin systems for membrane-associated interactions

  • Consider potential plant-specific cofactors or modifications

• In planta validation techniques:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Split luciferase complementation assays

Technical Considerations:

• Sample preparation critical factors:

  • Timing of harvest (developmental stage, diurnal regulation)

  • Tissue specificity (where BHLH149 is natively expressed)

  • Environmental conditions that may affect interactions

  • Crosslinking protocols to capture transient interactions

• Data analysis considerations:

  • Quantitative assessment of interaction strength

  • Competition assays to determine binding hierarchies

  • Domain mapping to identify interaction interfaces

  • Correlation with functional outcomes

These methodological considerations draw from successful approaches in plant protein interaction studies, where careful optimization of experimental conditions is crucial for detecting authentic interactions, particularly for transcription factors that often form dynamic regulatory complexes .

What are the current limitations of BHLH149 antibody research and potential future developments?

Current research with BHLH149 antibodies faces several limitations that future developments may address:

Current Technical Limitations:

• Limited validation for applications beyond ELISA and Western blotting

• Potential cross-reactivity with related BHLH family members

• Challenges in detecting low-abundance transcription factors in planta

• Polyclonal nature introducing batch-to-batch variability

• Limited sensitivity for detecting post-translational modifications

Promising Future Developments:

• Antibody engineering advancements:

  • Development of monoclonal antibodies for improved consistency

  • Recombinant antibody technology for renewable reagents

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Modification-specific antibodies for studying regulation

• Technological innovations:

  • Microfluidic antibody screening for improved specificity

  • AI-assisted epitope prediction for better immunogen design

  • CRISPR-based tagging for endogenous protein detection

  • Advances in proteomics requiring less starting material

• Integration with emerging methods:

  • Spatial proteomics for tissue-specific detection

  • Single-cell protein profiling technologies

  • High-throughput functional screening platforms

  • Improved computational prediction of antibody specificity

These future directions align with the broader trend in plant antibody research toward more reliable, specific, and versatile immunological tools that can support increasingly sophisticated systems biology approaches .

How do BHLH149 antibodies compare to other approaches for studying transcription factors in plants?

BHLH149 antibodies represent one of several complementary approaches for studying plant transcription factors, each with distinct advantages and limitations:

Comparative Analysis of Methods:

Integration Strategy:
The most effective research approach combines multiple methods, using antibodies for:

• Validating findings from other techniques

• Providing quantitative protein information

• Detecting specific protein modifications

• Examining endogenous expression patterns

This comparative perspective highlights the complementary nature of different research tools and emphasizes the continued value of well-validated antibodies in the plant molecular biology toolkit, particularly as we move toward more integrative understanding of complex biological systems .

What considerations should researchers keep in mind when interpreting results from BHLH149 antibody experiments?

When interpreting results from experiments utilizing BHLH149 antibodies, researchers should consider several important factors:

Technical Interpretation Considerations:

• Antibody validation thoroughness:

  • Extent of specificity testing performed

  • Controls included in the experiment

  • Cross-reactivity potential with related proteins

  • Batch-to-batch variability in polyclonal preparations

• Signal interpretation nuances:

  • Background signal versus specific detection

  • Quantitative limitations of Western blotting

  • Sensitivity thresholds for detecting low-abundance proteins

  • Potential for non-specific bands in complex plant extracts

Biological Context Factors:

• Physiological relevance:

  • Expression levels relative to natural abundance

  • Developmental timing and tissue specificity

  • Environmental conditions affecting expression

  • Relationship to known BHLH149 functions

• Regulatory considerations:

  • Post-translational modifications affecting detection

  • Protein complex formation masking epitopes

  • Protein turnover and stability factors

  • Subcellular compartmentalization affecting extraction

Data Integration Challenges:

• Correlation with other data types:

  • Consistency with transcript expression patterns

  • Agreement with genetic phenotypes

  • Alignment with predicted protein function

  • Integration with interaction network data

• Reproducibility assessment:

  • Biological versus technical replicates

  • Statistical analysis of quantitative data

  • Independent validation through complementary approaches

  • Publication of negative or inconsistent results

How might advances in plant antibody development impact broader plant biology research?

Advances in plant antibody technology, including improvements in BHLH149 antibodies, have the potential to significantly impact the broader field of plant biology:

Transformative Research Impacts:

• Systems biology advancement:

  • More accurate protein localization data for network modeling

  • Better quantification of protein dynamics in response to stimuli

  • Improved understanding of protein-protein interaction networks

  • Integration of protein-level data with other omics approaches

• Functional genomics acceleration:

  • Faster validation of gene function at protein level

  • More precise characterization of mutant phenotypes

  • Improved understanding of protein regulation mechanisms

  • Better tools for studying protein families and redundancy

Methodological Paradigm Shifts:

• Standardization improvements:

  • Development of community-wide antibody validation standards

  • Creation of comprehensive plant antibody repositories

  • Improved reproducibility across laboratories

  • Better research resource allocation through validated reagents

• Technological transfer:

  • Adaptation of medical antibody technologies to plant research

  • Development of plant-specific immunological techniques

  • Creation of multiplex detection systems for plant proteins

  • AI-assisted antibody design and validation

Future Research Directions:

• Addressing fundamental questions:

  • Protein complex composition in developmental pathways

  • Transcription factor dynamics during environmental responses

  • Post-translational modification networks in signaling

  • Spatial and temporal protein distribution in plant development

• Applied research acceleration:

  • Crop improvement through better protein characterization

  • Stress tolerance mechanisms at protein level

  • Plant-pathogen interaction studies with improved tools

  • Synthetic biology applications with better characterized components