BHLH145 Antibody

What is BHLH145 and why is it significant in plant research?

BHLH145 (Basic helix-loop-helix protein 145) is a transcription factor in Arabidopsis thaliana, also known as SACL2, EN131, or At5g50010 . It belongs to the bHLH family of transcription factors that regulate various developmental processes and stress responses in plants. The protein is primarily localized in the nucleus and functions as a DNA-binding transcription factor that regulates gene expression through interaction with specific DNA sequences.

The significance of BHLH145 lies in its role in controlling plant developmental processes. As a transcription factor, it likely influences gene expression networks that regulate specific physiological or developmental pathways in Arabidopsis. Understanding its function contributes to our knowledge of plant growth regulation, developmental transitions, and potentially stress responses.

What are the primary applications of BHLH145 antibodies in research?

BHLH145 antibodies serve multiple critical functions in plant molecular biology research:

• Western blotting: Detection of BHLH145 protein expression levels in different tissues or under various conditions at the expected molecular weight of approximately 35 kDa

• Immunohistochemistry: Visualization of protein localization within plant tissues and cells

• Chromatin immunoprecipitation (ChIP): Identification of genomic regions bound by BHLH145 to determine its direct transcriptional targets

• Co-immunoprecipitation: Investigation of protein-protein interactions involving BHLH145 in transcriptional complexes

• ELISA: Quantitative measurement of BHLH145 protein abundance across different experimental conditions

These applications allow researchers to investigate the expression patterns, subcellular localization, and functional interactions of BHLH145 in plant biology.

How should BHLH145 antibodies be stored and handled for optimal performance?

For maximum stability and activity of BHLH145 antibodies, the following storage and handling guidelines should be followed:

Reconstitution procedure:

• Briefly centrifuge the vial before opening to prevent loss of lyophilized material

• Reconstitute with 150 μl of sterile water for lyophilized antibodies

• Mix gently to ensure complete dissolution

Handling recommendations:

• Avoid repeated freeze-thaw cycles as they may denature the antibody and decrease activity

• Store in small aliquots after reconstitution to minimize freeze-thaw cycles

• Ship at 4°C and store immediately at recommended temperature upon receipt

• For working solutions, maintain sterile conditions to prevent contamination

What validation methods confirm the specificity of BHLH145 antibodies?

Validating BHLH145 antibody specificity is crucial for reliable experimental results. Standard validation methods include:

• Western blot analysis:

  • Using recombinant BHLH145 protein at various concentrations (2.5 ng, 10 ng, and 25 ng)

  • Should detect a single band at the expected molecular weight (35 kDa for native BHLH145 or 41 kDa for recombinant tagged protein)

  • Absence of non-specific bands indicates high specificity

• Peptide competition assay:

  • Pre-incubation of antibody with the immunizing peptide should block subsequent binding in Western blot or immunostaining

  • Similar to approaches used for other antibodies, where pre-incubation with specific peptides blocks antibody binding

• Knockout/knockdown controls:

  • Testing the antibody in BHLH145 knockout or knockdown plant lines

  • Should show reduced or absent signal compared to wild-type samples

• Cross-reactivity testing:

  • Evaluating antibody reactivity against related BHLH family members to ensure specificity

  • Important because the BHLH family contains multiple related proteins with similar structural domains

What sample preparation methods are optimal for BHLH145 detection in plant tissues?

Effective sample preparation is critical for successful detection of BHLH145 in plant tissues:

For Western blotting:

• Harvest fresh plant tissue and immediately freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining frozen state

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

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

• Collect supernatant and determine protein concentration

• Add sample buffer and heat at 95°C for 5 minutes

• Load 20-30 μg total protein per lane for SDS-PAGE separation

For immunohistochemistry:

• Fix plant tissues in 4% paraformaldehyde

• Embed in paraffin or prepare for cryosectioning

• Section tissues at 5-10 μm thickness

• Perform antigen retrieval using citrate buffer (pH 6.0)

• Block with 5% normal serum and proceed with antibody incubation

Nuclear protein enrichment (recommended for transcription factors):

• Isolate nuclei using sucrose gradient centrifugation

• Extract nuclear proteins using high-salt buffer

• This approach increases detection sensitivity by concentrating the nuclear-localized BHLH145 protein

How can BHLH145 antibodies be used to study protein-protein interactions in transcriptional complexes?

BHLH145 antibodies can be powerful tools for investigating protein-protein interactions within transcriptional complexes using the following approaches:

Co-immunoprecipitation (Co-IP):

• Prepare nuclear extracts from Arabidopsis tissues

• Incubate extracts with BHLH145 antibody coupled to protein A/G beads

• Wash to remove non-specific interactions

• Elute bound proteins and analyze by mass spectrometry or Western blot

• This technique can identify proteins that physically interact with BHLH145 in vivo

Proximity-dependent biotin identification (BioID):

• Generate transgenic plants expressing BHLH145 fused to a promiscuous biotin ligase

• Biotin treatment leads to biotinylation of proteins in close proximity to BHLH145

• Use BHLH145 antibodies to confirm expression of the fusion protein

• Isolate biotinylated proteins using streptavidin beads and identify by mass spectrometry

Chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS):

• Cross-link protein-DNA complexes in plant tissues

• Immunoprecipitate with BHLH145 antibody

• Identify co-precipitated proteins by mass spectrometry

• This approach can reveal proteins that interact with BHLH145 at chromatin sites

These methods are particularly valuable for understanding how BHLH145 functions within larger transcription factor complexes to regulate gene expression in plants.

What are the optimal conditions for chromatin immunoprecipitation (ChIP) using BHLH145 antibodies?

Optimizing ChIP protocols for BHLH145 requires careful consideration of several parameters:

Cross-linking conditions:

• Use 1% formaldehyde for 10-15 minutes at room temperature

• For weaker or transient interactions, consider dual cross-linking with disuccinimidyl glutarate (DSG) followed by formaldehyde

• Quench with 0.125 M glycine for 5 minutes

Chromatin fragmentation:

• Sonicate to generate fragments of 200-500 bp

• Optimize sonication conditions (amplitude, cycle number, duration) for plant tissues

• Verify fragment size by agarose gel electrophoresis

Immunoprecipitation parameters:

• Antibody amount: 2-5 μg per immunoprecipitation reaction

• Incubation: Overnight at 4°C with rotation

• Protein A/G beads: 30-50 μl of pre-blocked bead slurry

• Washing buffers: Use increasingly stringent wash buffers to minimize background

Controls for ChIP validation:

• Input DNA (non-immunoprecipitated chromatin)

• IgG control (non-specific antibody of same isotype)

• Positive control (known BHLH145 target regions)

• Negative control (genomic regions not expected to be bound by BHLH145)

qPCR primers for known or predicted BHLH145 binding sites:

• Design primers targeting E-box motifs (CANNTG) in promoter regions

• Include primers for housekeeping genes as negative controls

• Test primer efficiency using standard curves with input DNA

How do post-translational modifications affect BHLH145 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of BHLH145:

Potential effects on antibody binding:

• Phosphorylation may alter protein conformation or directly block epitopes, particularly if the epitope includes common phosphorylation sites

• Ubiquitination or SUMOylation can mask epitopes or create steric hindrance

• Glycosylation may prevent antibody access to epitopes in certain experimental conditions

Strategies to address PTM interference:

• Use multiple antibodies targeting different epitopes of BHLH145

• Phosphatase treatment of samples prior to immunoblotting to remove phosphorylation

• Deglycosylation enzymes to remove glycosyl groups that may interfere with antibody binding

• Denaturing conditions in Western blotting may expose epitopes that are masked in native conformations

PTM-specific antibodies:

• Consider developing modification-specific antibodies if particular PTMs of BHLH145 are functionally relevant

• These can be used to study the dynamics of BHLH145 modifications under different conditions

This understanding is especially important when conflicting results arise between different detection methods or experimental conditions.

What are the challenges in detecting BHLH145 in different plant tissues and developmental stages?

Detection of BHLH145 across tissues and developmental stages presents several technical challenges:

Variable expression levels:

• BHLH145 may be expressed at very low levels in certain tissues or developmental stages

• Enhanced detection methods may be required, such as signal amplification or enrichment of nuclear fractions

Tissue-specific interference:

• Plant tissues contain various compounds that can interfere with antibody binding:

  • Phenolic compounds may cross-link with proteins

  • Secondary metabolites may cause background fluorescence

  • Cell wall components may limit antibody penetration

Developmental regulation:

• BHLH145 expression and localization may change dramatically during development

• Timing of sample collection becomes critical for reproducible results

Recommended approaches for challenging samples:

• Tissue-specific extraction protocols optimized to remove interfering compounds

• Nuclear enrichment to concentrate BHLH145 protein from dilute samples

• Signal amplification techniques such as tyramide signal amplification for immunohistochemistry

• Tissue clearing methods for improved antibody penetration in whole-mount samples

• Developmental time course experiments to capture transient expression patterns

A systematic approach comparing detection methods across tissues and developmental stages can help establish optimal protocols for each specific research question.

How can researchers troubleshoot inconsistent results with BHLH145 antibodies?

When facing inconsistent results with BHLH145 antibodies, consider the following troubleshooting approaches:

Common problems and solutions:

Validation strategies:

• Positive controls: Include recombinant BHLH145 protein or overexpression samples

• Negative controls: Include knockdown/knockout samples if available

• Method comparison: Verify results using complementary methods (e.g., mass spectrometry)

• Antibody comparison: Test multiple antibodies targeting different BHLH145 epitopes

Documentation and reporting:

• Maintain detailed records of antibody lots, sample preparation methods, and experimental conditions

• Report all relevant details in publications to enhance reproducibility

Systematically addressing these factors can help identify the source of inconsistency and establish reliable protocols for BHLH145 detection.

How do BHLH145 antibodies compare to other detection methods for studying this transcription factor?

Understanding the comparative advantages of antibody-based detection versus other methods provides important context for experimental design:

Comparison of detection methods for BHLH145 analysis:

Complementary approaches:

• Combining antibody detection with transcript analysis provides correlation between mRNA and protein levels

• Verification of antibody results with tagged protein constructs can validate localization patterns

• Integration of ChIP-seq (antibody-based) with RNA-seq provides functional context for binding sites

The choice of detection method should be guided by the specific research question, available resources, and the strengths and limitations of each approach.

How can BHLH145 antibodies be incorporated into high-throughput or multiplexed experimental designs?

BHLH145 antibodies can be adapted for high-throughput and multiplexed studies using several advanced approaches:

Multiplexed immunoassays:

• Multiplex Western blotting:

  • Use antibodies from different species or isotypes

  • Label with distinct fluorophores for simultaneous detection

  • Allows co-detection of BHLH145 along with interaction partners or pathway components

• Protein microarrays:

  • Spot samples in array format for parallel analysis

  • Use BHLH145 antibodies to detect protein across multiple conditions

  • Enables screening of hundreds of samples simultaneously

High-content screening:

• Combine BHLH145 immunofluorescence with automated microscopy

• Quantify protein levels, subcellular localization, and co-localization with other factors

• Process thousands of cells or tissue sections in a single experiment

Single-cell applications:

• Adapt BHLH145 antibodies for mass cytometry (CyTOF) using metal-conjugated antibodies

• Enables single-cell analysis of BHLH145 in heterogeneous plant tissues

• Can be combined with multiple cellular markers for comprehensive phenotyping

Considerations for high-throughput optimization:

• Validate antibody performance in each specific application format

• Establish robust positive and negative controls for automated analysis

• Develop standardized protocols to ensure consistency across large sample sets

• Implement quality control metrics to identify technical artifacts

These approaches allow researchers to scale up BHLH145 studies and integrate them into systems biology frameworks.

What are the considerations for using BHLH145 antibodies in evolutionary studies across plant species?

When applying BHLH145 antibodies to study related proteins across different plant species, several factors must be considered:

Epitope conservation analysis:

• Align BHLH145 protein sequences from target species to assess epitope conservation

• Predict cross-reactivity based on sequence homology in the epitope region

• The central region of BHLH145 used as immunogen in some commercial antibodies shows variable conservation across plant species

Validation strategies for cross-species applications:

• Western blot validation in each target species

• Peptide competition assays to confirm specificity

• Recombinant protein controls from each species of interest

• Immunoprecipitation followed by mass spectrometry to confirm target identity

Evolutionary considerations:

• BHLH proteins have undergone significant diversification during plant evolution

• Functional conservation may not correlate with sequence conservation

• Paralogous BHLH proteins in different species may have divergent functions

Recommended approach for evolutionary studies:

This careful approach allows meaningful cross-species comparisons while avoiding artifacts from variable antibody reactivity.

What emerging technologies might enhance the utility of BHLH145 antibodies in plant research?

Several cutting-edge technologies are poised to expand the applications of BHLH145 antibodies:

Proximity-dependent labeling technologies:

• TurboID or miniTurbo fusions with BHLH145 for rapid biotin labeling of proximal proteins

• Integration with antibody validation to confirm proper fusion protein function

• Enables mapping of dynamic protein interaction networks in living plant cells

Super-resolution microscopy:

• Antibody-based detection of BHLH145 with techniques like STORM, PALM, or STED

• Achieves nanoscale resolution of protein localization beyond the diffraction limit

• Reveals detailed nuclear distribution patterns and co-localization with chromatin features

In situ protein analysis:

• Antibody-based detection combined with in situ sequencing or spatial transcriptomics

• Correlates BHLH145 protein distribution with gene expression patterns

• Provides spatial context for transcription factor function in complex tissues

CRISPR-based approaches:

• CRISPR-based tagging of endogenous BHLH145 for antibody-free detection

• Validation using existing antibodies ensures accurate representation of native protein

• Enables live-cell imaging while maintaining physiological expression levels

Single-molecule tracking:

• Fluorophore-conjugated antibody fragments for tracking individual BHLH145 molecules

• Reveals dynamics of DNA binding, residence time, and mobility within the nucleus

• Provides mechanistic insights into transcription factor function

These technologies represent promising directions for advanced BHLH145 research, potentially revealing new aspects of its function and regulation in plant biology.

How can researchers contribute to improving BHLH145 antibody resources for the scientific community?

Researchers can enhance the quality and availability of BHLH145 antibody resources through several collaborative approaches:

Standardized validation and reporting:

• Implement comprehensive validation protocols for BHLH145 antibodies

• Document detailed methods, including:

  • Exact experimental conditions

  • Cell/tissue types tested

  • Positive and negative controls used

  • Observed limitations or cross-reactivity

• Share validation data in public repositories or as supplementary material in publications

Resource development:

• Generate and characterize monoclonal antibodies against different BHLH145 epitopes

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

• Create knockout/knockdown validation lines and share as community resources

Data sharing platforms:

• Contribute to antibody validation databases like Antibodypedia or CiteAb

• Share protocols on platforms like protocols.io

• Deposit detailed methods in repositories specific to plant research

Community standards:

• Participate in establishing minimum validation standards for plant antibodies

• Advocate for transparent reporting of antibody validation in publications

• Support initiatives for independent antibody validation

By contributing to these efforts, researchers can collectively improve the reliability and reproducibility of BHLH145 research and accelerate progress in understanding this important transcription factor.