BHLH140 Antibody

What is BHLH140 and what cellular processes does it regulate?

BHLH140 (Basic Helix-Loop-Helix protein 140) is a transcription factor belonging to the bHLH family in Arabidopsis thaliana. It functions as a DNA-binding protein that regulates gene expression by recognizing specific DNA sequences such as E-box motifs (CANNTG) . As a member of the bHLH transcription factor family, it likely plays roles in developmental processes, stress responses, or metabolism regulation.

The bHLH domain consists of approximately 60 amino acids with two functionally distinct regions: the basic region at the N-terminal end involved in DNA binding, and the HLH region at the C-terminal end that functions as a dimerization domain . BHLH140 is encoded by the gene At5g01310 and is also known as EN122.

How is BHLH140 structurally and functionally related to other bHLH transcription factors?

BHLH140 shares the characteristic domain architecture of the bHLH family, featuring:

• A basic DNA-binding region composed of approximately 15 amino acids with numerous basic residues

• A helix-loop-helix region consisting of two amphipathic α-helices separated by a loop region

In Arabidopsis, comprehensive phylogenetic analysis has revealed 147-152 bHLH proteins that can be classified into 21 subfamilies based on their sequence similarities . These proteins often function as homo- or heterodimers, with the HLH region mediating protein-protein interactions and the basic region recognizing specific DNA sequences. The dimerization capacity allows for combinatorial interactions that expand their regulatory potential in controlling diverse transcriptional programs .

The predicted subcellular localization of BHLH140 is the nucleus, consistent with its role as a transcription factor.

What are the recommended applications for BHLH140 antibody?

Based on product information, the BHLH140 antibody is primarily validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blot (WB)

These techniques are fundamental for detecting and quantifying BHLH140 protein in various experimental contexts. The antibody is designed to specifically recognize Arabidopsis thaliana BHLH140 protein and can be used to study its expression patterns, protein levels, and potential post-translational modifications.

For optimal results, researchers should design experiments considering that:

• BHLH140 antibody is polyclonal, raised in rabbit against recombinant Arabidopsis thaliana BHLH140 protein

• The storage buffer contains 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4

• It's recommended to store the antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles

How should I optimize Western blot conditions for BHLH140 detection?

For successful Western blot experiments with BHLH140 antibody:

• Sample preparation:

  • Extract nuclear proteins since BHLH140 is primarily localized in the nucleus

  • Include protease inhibitors to prevent degradation of the target protein

  • Use appropriate lysis buffers optimized for nuclear proteins

• Gel electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels to effectively resolve proteins in the expected molecular weight range

  • Load sufficient protein (20-50 μg of total protein) to ensure detection of low-abundance transcription factors

• Transfer conditions:

  • Semi-dry or wet transfer systems can be used with optimization for nuclear proteins

  • PVDF membranes may provide better results than nitrocellulose for nuclear transcription factors

• Antibody dilution:

  • For Western blot applications, start with a dilution range of 0.2-0.5 μg/ml, which is recommended for mouse-derived antibodies

  • For rabbit-derived antibodies like BHLH140, consider using a slightly lower concentration (20-50 ng/ml)

  • Optimize through titration experiments if initial results are unsatisfactory

• Detection system:

  • Both chemiluminescence and fluorescence-based detection systems are compatible

  • For low abundance targets, enhanced chemiluminescence may provide better sensitivity

How can I improve specificity when using BHLH140 antibody in plant tissue samples?

When working with plant tissues, several strategies can enhance antibody specificity:

• Tissue preparation:

  • Use young, actively growing tissues where transcription factors are more abundantly expressed

  • For Arabidopsis, consider using seedlings or specific tissues known to express BHLH140

  • Fresh tissue typically yields better results than stored samples

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat dry milk, commercial blockers)

  • Plant samples may benefit from 5% BSA in TBS-T rather than milk-based blockers

  • Include 0.1% Tween-20 in wash buffers to reduce background

• Cross-reactivity minimization:

  • Pre-absorb the antibody with plant extract from knockout or knockdown lines lacking BHLH140

  • Include competing proteins in the antibody diluent to reduce non-specific binding

  • Consider longer, more frequent washing steps (4-5 washes of 10 minutes each)

• Controls to include:

  • Positive control: Overexpression line of BHLH140

  • Negative control: bhlh140 knockout/knockdown line

  • Technical control: Primary antibody omission

  • Specificity control: Preincubation of antibody with excess antigen

What are common issues with BHLH140 antibody detection and how can they be resolved?

For troubleshooting approaches specific to transcription factor detection:

• Nuclear enrichment protocols can significantly improve detection sensitivity

• Consider crosslinking before extraction to preserve protein-DNA interactions

• For developmental studies, carefully stage plant materials as transcription factor expression can be highly temporal

How can BHLH140 antibody be utilized in chromatin immunoprecipitation (ChIP) experiments?

Though not explicitly listed among the validated applications for BHLH140 antibody, researchers interested in identifying BHLH140 binding sites can adapt ChIP protocols based on approaches used for other bHLH transcription factors:

• Experimental design considerations:

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

  • Chromatin fragmentation: Optimize sonication conditions to achieve fragments of 200-500 bp

  • Immunoprecipitation: Use 2-5 μg of antibody per ChIP reaction

  • Include appropriate controls: IgG control, input control, and positive control regions

• Protocol adaptations for plant tissues:

  • Consider vacuum infiltration for efficient crosslinking

  • Use glass beads or specialized disruption methods for plant cell wall lysis

  • Include plant-specific protease inhibitors in all buffers

  • Pre-clear lysates with protein A/G beads to reduce background

• Data analysis approach:

  • Focus on E-box motifs (CANNTG) and particularly G-box motifs (CACGTG) which are commonly bound by bHLH transcription factors

  • Perform motif enrichment analysis to identify BHLH140-specific binding preferences

  • Consider the effect of potential heterodimerization with other bHLH proteins on binding specificity

Based on research with other bHLH transcription factors, BHLH140 likely regulates genes by binding to E-box elements in their promoters, similar to the characterized interactions of other family members .

How can I investigate BHLH140 protein-protein interactions to understand its transcriptional network?

To investigate BHLH140 interaction partners:

• Co-immunoprecipitation (Co-IP) approach:

  • Use BHLH140 antibody to immunoprecipitate the protein complex from plant nuclear extracts

  • Mild lysis conditions are essential to preserve protein-protein interactions

  • Identify interacting partners through mass spectrometry analysis of co-precipitated proteins

  • Validate interactions through reciprocal Co-IP or alternative methods

• Yeast two-hybrid screening:

  • Clone the BHLH140 coding sequence into appropriate bait vectors

  • Screen against Arabidopsis cDNA libraries or specific candidate interactors

  • Focus on other transcription factors, particularly other bHLH family members

  • Validate interactions through BiFC (Bimolecular Fluorescence Complementation) in planta

• Investigating heterodimerization patterns:

  • bHLH proteins frequently form heterodimers to expand their regulatory potential

  • Evidence from other bHLH proteins suggests that dimerization patterns significantly impact DNA binding specificity and biological function

  • For instance, PIF3 and PIF4, two related phytochrome-interacting bHLH members in Arabidopsis, can form both homodimers and heterodimers, with all three configurations binding to G-box DNA sequences

How can I differentiate between direct and indirect regulatory targets of BHLH140?

To establish direct regulatory relationships:

• Integrated approach combining:

  • ChIP-seq to identify genome-wide binding sites

  • RNA-seq to determine differentially expressed genes in bhlh140 mutants versus wild-type

  • The intersection represents potential direct targets (both bound and regulated)

• Validation strategies:

  • Perform ChIP-qPCR on selected targets to confirm binding

  • Use reporter gene assays with wild-type and mutated E-box elements from target promoters

  • Employ inducible systems (e.g., glucocorticoid-inducible or estradiol-inducible BHLH140) combined with cycloheximide treatment to identify primary response genes

• Data interpretation framework:

  • Consider that transcription factors often bind many sites without functional regulation

  • The absence of an expression change doesn't necessarily indicate lack of regulation (redundancy may mask effects)

  • Binding site location relative to transcription start sites can provide insights into regulatory mechanisms

What approaches can reveal the functional role of BHLH140 in development and stress responses?

To elucidate BHLH140 function in plants:

• Genetic analysis approach:

  • Generate and characterize knockout/knockdown lines (T-DNA insertion, CRISPR-Cas9, RNAi)

  • Create overexpression lines under constitutive and tissue-specific promoters

  • Analyze phenotypes under normal growth conditions and various stresses

  • Perform complementation experiments to confirm phenotype-genotype relationships

• Expression pattern analysis:

  • Use quantitative real-time PCR (RT-qPCR) to measure BHLH140 expression across tissues and conditions

  • Generate promoter-reporter fusions (BHLH140pro:GUS or BHLH140pro:GFP) to visualize expression patterns

  • Standard qPCR conditions: 95°C for 30s, followed by 40 cycles of 95°C for 10s and 60°C for 30s

  • Analyze gene expression levels using the 2^-ΔΔCT method with appropriate reference genes

• Functional genomics integration:

  • Perform transcriptome analysis (RNA-seq) comparing wild-type and mutant plants

  • Conduct Gene Ontology enrichment analysis of differentially expressed genes

  • Map the results onto known biological pathways to identify processes regulated by BHLH140

  • Consider chromatin dynamics, as bHLH transcription factors can cooperate with chromatin remodelers to regulate cell fate decisions

How conserved is BHLH140 across plant species and what does this suggest about its function?

Understanding the evolutionary conservation of BHLH140:

• Comparative genomics approach:

  • Identify orthologs of BHLH140 in other plant species through reciprocal BLAST searches

  • Perform multiple sequence alignments focusing on the bHLH domain and other functional regions

  • Construct phylogenetic trees to determine evolutionary relationships with other bHLH proteins

  • The bHLH family in Arabidopsis has been classified into 21 subfamilies based on phylogenetic analysis

• Structural conservation analysis:

  • Examine conservation of key functional residues, particularly in the DNA-binding basic region

  • Analyze conservation of protein-interaction surfaces in the HLH region

  • Investigate whether domain architecture is preserved across species

  • Predict functional importance based on evolutionary constraint patterns

• Functional prediction framework:

  • Highly conserved orthologs likely maintain similar functions across species

  • Differences in conservation may indicate species-specific adaptations

  • Compare expression patterns of orthologs in different plant species to identify conserved regulatory modules

  • Cross-species complementation experiments can provide evidence for functional conservation

How does the DNA binding specificity of BHLH140 compare to other family members?

To characterize BHLH140 DNA binding preferences:

• Sequence-based prediction:

  • Analyze the basic region sequence of BHLH140 to predict its DNA-binding specificity

  • Compare with characterized bHLH proteins grouped according to DNA binding preferences:

    • Group A: Binds to E-box variant CAGCTG

    • Group B: Binds to G-box (CACGTG)

    • Group C: Binds to non-E-box sequences (NACGTG or NGCGTG)

• Experimental determination approaches:

  • Perform EMSA (Electrophoretic Mobility Shift Assay) with recombinant BHLH140 and labeled DNA probes

  • Conduct protein-binding microarrays to determine sequence preferences systematically

  • Utilize ChIP-seq data to identify enriched motifs in vivo

• Heterodimerization effects on binding specificity:

  • bHLH proteins often form both homodimers and heterodimers with differential binding preferences

  • The selection of dimerization partners can significantly alter DNA sequence recognition

  • For example, research on PIF3 and PIF4 has shown that both homodimers and heterodimers can bind to G-box DNA sequences, suggesting potential combinatorial regulation

  • These different dimeric configurations may regulate distinct sets of target genes

Through this comprehensive characterization of DNA binding preferences, researchers can better understand the regulatory networks controlled by BHLH140 and its position within the broader transcriptional landscape of Arabidopsis.