BHLH71 Antibody

What is BHLH71 and what is its molecular function in plants?

BHLH71 belongs to the basic helix-loop-helix (bHLH) family of transcription factors that regulate various biological processes in plants. Recent research has identified BHLH71-like as a positive regulator of carotenoid biosynthesis in pepper plants. Unlike other bHLH transcription factors that typically negatively regulate carotenoid biosynthesis genes, BHLH71-like shows a positive correlation with light intensity and functions as an activator of carotenoid biosynthesis .

The protein localizes to the nucleus, consistent with its role as a transcription factor, and can bind to G-box elements in the promoters of target genes such as CaVDE (violaxanthin de-epoxidase) . When expressed at higher levels under high light intensity, BHLH71-like promotes the accumulation of yellow carotenoid pigments like zeaxanthin and antheraxanthin, contributing to a yellowing phenotype in mutant plants . This function suggests BHLH71's importance in light adaptation mechanisms and photoprotection in plants.

What are the primary applications of BHLH71 antibodies in plant molecular biology research?

BHLH71 antibodies serve multiple critical functions in plant molecular biology research, including:

• Protein localization studies: Antibodies can verify the nuclear localization of BHLH71 in different plant tissues and under various light conditions, complementing fluorescent protein fusion approaches .

• Protein expression analysis: Western blotting with BHLH71 antibodies allows quantification of protein levels across different tissues or in response to environmental stimuli like varying light intensities.

• Chromatin immunoprecipitation (ChIP): BHLH71 antibodies can identify DNA binding sites and target genes regulated by this transcription factor, similar to the demonstrated binding of BHLH71-like to the CaVDE promoter .

• Protein-protein interaction studies: Co-immunoprecipitation with BHLH71 antibodies can help identify interacting partners in transcriptional complexes.

• Tissue-specific expression profiling: Immunohistochemistry using BHLH71 antibodies can reveal the spatial distribution of the protein across different plant tissues, complementing the gene expression analyses showing highest expression in leaf tissues followed by fruit and flower tissues .

How can I validate the specificity of a BHLH71 antibody?

Validating antibody specificity is crucial for reliable results, particularly for transcription factor families with high sequence homology like bHLHs. A comprehensive validation approach should include:

• Western blot analysis using recombinant BHLH71 protein as a positive control

• Testing on wild-type plant extracts versus BHLH71 knockdown/knockout lines (similar to the VIGS-silenced lines described in the research)

• Peptide competition assays to confirm binding specificity

• Cross-reactivity testing against closely related bHLH family members, particularly important as the search results mention multiple bHLH transcription factors (36 with FPKM values >10) expressed in pepper plants

• Testing in different plant tissues with known expression levels of BHLH71, such as higher expression in leaves compared to roots and stems

Specificity validation is especially important given that BHLH71 belongs to a large family of transcription factors with 36 different bHLH transcription factors identified in pepper transcriptomes with significant expression levels .

What is the optimal protocol for subcellular localization studies using BHLH71 antibodies?

For effective subcellular localization of BHLH71, researchers should consider a dual approach combining antibody-based immunofluorescence with complementary techniques:

• Primary method: Immunofluorescence microscopy

  • Fix plant tissues with 4% paraformaldehyde

  • Permeabilize cell walls and membranes with an appropriate detergent

  • Block with BSA or normal serum

  • Incubate with validated BHLH71 primary antibody

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain with DAPI to visualize nuclei

  • Image using confocal microscopy

• Complementary approach: GFP fusion validation system

  • Similar to the approach used for BHLH71-like, where researchers fused the coding sequence without a stop codon to GFP in the pCAMBIA1300 vector

  • Co-express with a nuclear marker like HY5-mCherry

  • Visualize using confocal laser scanning microscopy to confirm nuclear localization

The combination of these approaches provides stronger evidence of authentic localization patterns and helps control for potential artifacts in either method. The search results demonstrate that BHLH71-like showed clear nuclear localization when fused to GFP and co-expressed with the nuclear marker HY5-mCherry in Nicotiana benthamiana leaves .

How can I optimize ChIP protocols using BHLH71 antibodies to identify DNA binding targets?

Optimizing ChIP protocols for BHLH71 requires careful attention to several key aspects:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (1-3%) and incubation times (10-20 minutes)

  • For plant tissues, vacuum infiltration may improve crosslinking efficiency

  • Consider dual crosslinking with disuccinimidyl glutarate followed by formaldehyde for stronger protein-DNA associations

• Chromatin fragmentation:

  • Optimize sonication conditions for consistent DNA fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Pre-clear chromatin with protein A/G beads

  • Use optimized antibody concentration (typically 1-5 μg per IP reaction)

  • Include negative controls: IgG isotype control and input chromatin

• Validation with known targets:

  • Design primers for the G-box elements in the CaVDE promoter as a positive control, since BHLH71-like has been shown to bind this region

  • Include primers for non-target regions as negative controls

• Data analysis:

  • Quantify enrichment using qPCR relative to input and IgG controls

  • For genome-wide analysis, proceed with ChIP-seq library preparation

The yeast one-hybrid assays and dual luciferase reporter assays used in the BHLH71-like research provide valuable information about potential DNA binding sites, particularly the binding to the CaVDE promoter, which contains G-box elements . This information can serve as a starting point for designing ChIP experiments.

What are the best extraction methods to preserve BHLH71 protein integrity for antibody detection?

Extracting nuclear transcription factors like BHLH71 while preserving their integrity requires specialized approaches:

• Nuclear extraction protocol:

  • Homogenize fresh tissue in nuclear isolation buffer (20 mM Tris-HCl pH 7.4, 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl₂, 250 mM sucrose)

  • Add protease inhibitors (PMSF, leupeptin, aprotinin) and phosphatase inhibitors if studying phosphorylation states

  • Include reducing agents (DTT or β-mercaptoethanol) to preserve disulfide bonds

  • Filter through miracloth and centrifuge at 1,000g for 10 minutes at 4°C

  • Resuspend the nuclear pellet in protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate)

• Critical considerations:

  • Maintain cold temperature throughout extraction (4°C)

  • Optimize detergent concentration to solubilize nuclear membranes without denaturing proteins

  • Include 10-15% glycerol in final extracts to stabilize protein structure

  • Use freshly prepared samples whenever possible, or flash-freeze in liquid nitrogen

• Quality control:

  • Verify nuclear enrichment by immunoblotting for nuclear markers (histones)

  • Check for cytoplasmic contamination using markers like tubulin

  • Assess protein integrity by SDS-PAGE and Coomassie staining

The experimenters in the referenced study worked with both full-length protein for localization studies and the BHLH71-like coding sequence for yeast one-hybrid assays, suggesting that preserving the functional domains is essential for maintaining protein activity .

How can BHLH71 antibodies be used to study protein-protein interactions in transcriptional complexes?

BHLH71 antibodies can be instrumental in studying protein-protein interactions through several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare nuclear extracts under non-denaturing conditions

  • Immunoprecipitate BHLH71 using specific antibodies

  • Analyze co-precipitated proteins by mass spectrometry or western blotting for suspected partners

  • Include appropriate controls: IgG isotype control, reverse Co-IP with antibodies against suspected partners

• Proximity ligation assay (PLA):

  • Useful for detecting in situ protein-protein interactions

  • Fix and permeabilize plant tissues

  • Incubate with primary antibodies against BHLH71 and a potential interacting protein

  • Apply PLA probes, ligase, and polymerase according to manufacturer's protocol

  • Fluorescent signals indicate proximity (<40 nm) between proteins

• Bimolecular fluorescence complementation (BiFC) as a complementary method:

  • Similar to the experimental approach used to verify protein-protein interactions

  • Split YFP or GFP fragments are fused to BHLH71 and potential interacting proteins

  • Co-expression in plant cells results in fluorescence only if proteins interact

• Yeast two-hybrid screening:

  • Use BHLH71 as bait to screen for interacting proteins

  • Validate interactions using antibody-based methods above

These approaches would help identify proteins that might form complexes with BHLH71 to regulate carotenoid biosynthesis genes, similar to how the researchers demonstrated BHLH71-like binding to the CaVDE promoter .

What approaches can be used to study posttranslational modifications of BHLH71 with antibodies?

Studying posttranslational modifications (PTMs) of BHLH71 requires specialized antibodies and techniques:

• Modification-specific antibodies:

  • Use phospho-specific, acetylation-specific, or other PTM-specific antibodies against BHLH71

  • If commercial antibodies are unavailable, consider custom antibody development against predicted modification sites

  • Validate specificity using in vitro modified recombinant BHLH71

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate BHLH71 using specific antibodies

  • Perform tryptic digestion of the immunoprecipitated protein

  • Analyze peptides by LC-MS/MS to identify PTMs

  • Compare modification patterns between different experimental conditions (e.g., high vs. low light intensity)

• Western blotting with mobility shift analysis:

  • Some PTMs (particularly phosphorylation) cause mobility shifts on SDS-PAGE

  • Compare migration patterns before and after treatment with phosphatases or other modification-removing enzymes

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Compare spot patterns to identify modified forms of BHLH71

  • Excise spots for MS confirmation of modifications

Understanding PTMs could provide insight into how light intensity regulates BHLH71 activity, as the research showed that BHLH71-like expression levels positively correlated with light intensity in the yl1 mutant . PTMs might be an additional regulatory layer beyond transcriptional control.

How can I design experiments to study the dynamic interaction between BHLH71 and its target promoters under different light conditions?

To study dynamic interactions between BHLH71 and its target promoters under varying light conditions, consider these experimental approaches:

• Time-course ChIP experiments:

  • Subject plants to different light intensities (e.g., low, medium, high) as in the referenced study (50, 200, 500 μmol/m²/s)

  • Collect samples at multiple time points after light treatment

  • Perform ChIP with BHLH71 antibodies

  • Quantify binding to target promoters (e.g., CaVDE) by qPCR

  • Plot temporal binding profiles under different light conditions

• Dual crosslinking ChIP for transient interactions:

  • Use protein-protein crosslinker (DSG) followed by formaldehyde

  • This approach captures more transient or weak DNA-protein interactions

• ChIP-seq combined with RNA-seq:

  • Perform parallel ChIP-seq and RNA-seq analyses under different light conditions

  • Correlate BHLH71 binding patterns with changes in gene expression

  • Identify direct vs. indirect regulatory targets

• In vivo footprinting:

  • Use ligation-mediated PCR or other footprinting methods

  • Detect changes in chromatin accessibility at BHLH71 binding sites under different light conditions

• Live-cell imaging with fluorescently tagged BHLH71:

  • Create fluorescent protein fusions similar to the GFP-BHLH71-like construct used for localization studies

  • Use fluorescence recovery after photobleaching (FRAP) to measure binding dynamics

  • Compare mobility/residence time under different light conditions

These approaches would extend the findings that BHLH71-like can bind to the CaVDE promoter (as shown by yeast one-hybrid and dual luciferase assays) by examining the dynamics of this interaction under different physiological conditions.

How do I troubleshoot weak or inconsistent signals when using BHLH71 antibodies in Western blots?

Inconsistent or weak signals are common challenges when working with transcription factor antibodies. Here's a systematic troubleshooting approach:

• Sample preparation issues:

  • Ensure complete extraction of nuclear proteins using appropriate nuclear extraction buffers

  • Add protease inhibitors immediately before extraction

  • Optimize protein loading (10-30 μg for nuclear extracts)

  • Verify protein integrity by Ponceau S staining of membranes

• Antibody-related factors:

  • Titrate antibody concentration (typically 1:500 to 1:5000 dilution)

  • Extend primary antibody incubation (overnight at 4°C)

  • Try different secondary antibodies or detection systems

  • Consider using antibody enhancer solutions

• Protocol optimization:

  • Test different blocking agents (5% milk vs. 3-5% BSA)

  • Optimize transfer conditions for high molecular weight proteins

  • Increase exposure time during detection

  • Try different membrane types (PVDF may give better results than nitrocellulose for some applications)

• Controls to include:

  • Positive control: extracts from tissues with known high BHLH71 expression (leaf tissue showed highest expression of BHLH71-like )

  • Negative control: extracts from BHLH71-silenced plants (similar to the VIGS approach used for BHLH71-like )

  • Loading control: nuclear protein (histone) to normalize signals

• Special considerations for BHLH71:

  • Given its role as a transcription factor, expression levels may be relatively low

  • Consider using signal amplification methods like enhanced chemiluminescence substrates

The research on BHLH71-like demonstrated successful detection of gene expression changes using qRT-PCR , which might be a complementary approach to verify protein-level findings.

How can I interpret contradictory results between transcript and protein levels of BHLH71?

Discrepancies between transcript and protein levels are common in biological systems and can provide important insights:

• Possible mechanistic explanations:

  • Post-transcriptional regulation (miRNAs, RNA stability)

  • Translational control mechanisms

  • Post-translational modifications affecting protein stability

  • Protein degradation pathways (ubiquitin-proteasome system)

• Analytical approach:

  • Quantify both transcript (qRT-PCR) and protein levels (Western blot with BHLH71 antibodies)

  • Include multiple time points to detect temporal shifts between mRNA and protein changes

  • Measure protein half-life using cycloheximide chase experiments

  • Assess ubiquitination status by immunoprecipitation followed by ubiquitin blotting

• Data integration strategy:

  • Plot transcript vs. protein levels across conditions and time points

  • Calculate correlation coefficients to quantify relationship

  • Determine lag time between transcript and protein changes

  • Create mathematical models to explain the relationship

• Biological interpretation:

  • In stress responses like high light conditions, rapid protein regulation often precedes transcriptional changes

  • For transcription factors like BHLH71, protein activity may be primarily regulated post-translationally while maintaining stable transcript levels

In the BHLH71-like study, researchers observed that expression levels were positively correlated with light intensity in the yl1 mutant but remained stable in wild-type plants , suggesting complex regulatory mechanisms that may differ between transcript and protein levels.

What statistical approaches are appropriate for analyzing ChIP data obtained using BHLH71 antibodies?

• ChIP-qPCR analysis:

  • Calculate percent input or fold enrichment over IgG control

  • Use ANOVA with post-hoc tests for comparing multiple conditions

  • Apply non-parametric tests (Mann-Whitney U or Kruskal-Wallis) if data doesn't meet normality assumptions

  • Include biological replicates (minimum n=3) for statistical power

• ChIP-seq data analysis:

  • Quality control: assess sequence quality, adapter contamination, and duplicate rates

  • Peak calling: use MACS2, HOMER, or other specialized algorithms with appropriate p-value thresholds

  • Differential binding analysis: DiffBind or similar tools for comparing binding across conditions

  • Multiple testing correction: apply Benjamini-Hochberg FDR method

• Integrative analysis:

  • Motif enrichment: analyze sequences under peaks for G-box elements and other motifs

  • Pathway analysis: determine if binding sites are enriched near genes in particular pathways

  • Integration with expression data: correlate binding with RNA-seq or microarray data

• Visualization and reporting:

  • Generate genome browser tracks showing binding intensity

  • Create heatmaps of binding strength across different conditions

  • Use volcano plots to display statistical significance vs. fold change in differential binding

When interpreting ChIP data for BHLH71, consider its demonstrated ability to bind G-box elements in target promoters, as shown for the CaVDE promoter in yeast one-hybrid and dual luciferase assays .

How can BHLH71 antibodies contribute to studying the role of this transcription factor in plant stress responses?

BHLH71 antibodies can enable several research directions for understanding plant stress responses:

• Stress-induced changes in BHLH71 localization and abundance:

  • Use immunolocalization and Western blotting to track BHLH71 under various stresses

  • Compare responses to different light intensities, as BHLH71-like expression correlated with light intensity

  • Examine other abiotic stresses (temperature, drought, salt) that might interact with light response pathways

• Chromatin dynamics during stress adaptation:

  • Perform ChIP-seq under normal and stress conditions to identify stress-specific binding sites

  • Compare binding patterns across stress types to identify common and unique targets

  • Integrate with chromatin accessibility data (ATAC-seq) to understand stress-induced chromatin remodeling

• Protein interaction networks in stress signaling:

  • Use co-immunoprecipitation with BHLH71 antibodies followed by mass spectrometry

  • Compare interactomes under different stress conditions

  • Build stress-specific protein interaction networks

• Post-translational modifications in stress signaling:

  • Develop or use modification-specific antibodies

  • Track PTM patterns under different stress conditions

  • Correlate modifications with transcriptional activity and target selectivity

• Tissue-specific stress responses:

  • Use immunohistochemistry to map BHLH71 expression across tissues during stress

  • Correlate with physiological responses and stress sensitivity

The research on BHLH71-like showed its important role in light stress responses, affecting the accumulation of photoprotective carotenoids like zeaxanthin and antheraxanthin , suggesting that BHLH71 may be a key player in multiple stress response pathways.

What are the most promising approaches for studying the evolutionary conservation of BHLH71 function across plant species?

Studying evolutionary conservation of BHLH71 across plant species requires integrative approaches:

• Comparative antibody-based studies:

  • Test cross-reactivity of BHLH71 antibodies across related plant species

  • Compare expression patterns in homologous tissues

  • Assess subcellular localization conservation

• Functional conservation analysis:

  • Identify BHLH71 homologs across species using phylogenetic analysis

  • Perform ChIP with BHLH71 antibodies in multiple species

  • Compare binding motifs and target genes

  • Analyze conservation of protein-protein interactions

• Domain-specific antibody approaches:

  • Develop antibodies against highly conserved domains

  • Use these to study functionally important regions across species

  • Compare modification patterns of conserved residues

• Complementation experiments:

  • Express BHLH71 from different species in the pepper yl1 mutant background

  • Use antibodies to confirm expression

  • Assess phenotypic rescue of the yellowing phenotype under high light

• Evolutionary rate analysis:

  • Compare sequence conservation with functional conservation

  • Identify rapidly or slowly evolving domains

  • Correlate with antibody epitope regions for interpretation of cross-reactivity data

The research on BHLH71-like in pepper plants revealed its function as a positive regulator of carotenoid biosynthesis , which contrasts with typical bHLH transcription factors that negatively regulate carotenoid biosynthesis genes . This suggests interesting evolutionary divergence in function that merits comparative study across species.