BHLH147 Antibody

Basic Research Questions

• What is the BHLH147 protein and why is it important in plant research?

  BHLH147 (UniProt: Q9LSN7) is a transcription factor belonging to the basic helix-loop-helix (bHLH) family in Arabidopsis thaliana (Mouse-ear cress). This protein plays regulatory roles in plant development, stress responses, and metabolic pathways. The bHLH transcription factors are characterized by two functionally distinct regions: the basic region involved in DNA binding and the helix-loop-helix region mediating protein-protein interactions. Research on BHLH147 contributes to our understanding of transcriptional regulation in plants, particularly in stress response mechanisms and developmental processes .

• What are the key specifications of the BHLH147 Antibody?

  The BHLH147 Antibody has the following technical specifications:

  Parameter	Specification
  Product Code	CSB-PA868010XA01DOA
  Host/Source	Rabbit
  Clonality	Polyclonal
  Immunogen	Recombinant Arabidopsis thaliana BHLH147 protein
  Purification Method	Antigen Affinity Purified
  Applications	ELISA, Western Blotting
  Species Reactivity	Arabidopsis thaliana
  Isotype	IgG
  Storage Conditions	-20°C or -80°C (avoid repeated freeze-thaw cycles)
  Storage Buffer	0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

  This antibody has been specifically designed for plant research applications and undergoes antigen affinity purification to ensure high specificity in experimental contexts .

• What experimental applications is the BHLH147 Antibody validated for?

  The BHLH147 Antibody has been validated for the following applications:

  • ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of BHLH147 protein levels in plant tissue extracts. This application is particularly useful for screening multiple samples and comparative expression studies.

  • Western Blotting (WB): For identification and semi-quantitative analysis of BHLH147 protein expression in plant lysates. This technique allows researchers to determine protein size, verify antibody specificity, and monitor expression changes under various conditions.

  When designing experiments, researchers should optimize antibody concentration for each specific application and include appropriate positive and negative controls to ensure reliable results .

Intermediate Research Questions

• What are the recommended protocols for optimizing Western blotting with BHLH147 Antibody?

  Optimizing Western blot protocols for plant transcription factors like BHLH147 requires specific considerations:

  1. Sample Preparation:

     • Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail

     • For nuclear proteins like BHLH147, include a nuclear extraction step before general protein extraction

     • Use liquid nitrogen grinding followed by buffer extraction for most efficient protein recovery from plant tissues

  2. Electrophoresis and Transfer Parameters:

     • Use 10-12% SDS-PAGE gels for optimal resolution

     • Transfer proteins to PVDF membranes (rather than nitrocellulose) for better protein retention

     • Apply 100V for 60-90 minutes during transfer with cooling system

  3. Blocking and Antibody Incubation:

     • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

     • Dilute primary BHLH147 Antibody 1:500 to 1:1000 in blocking solution

     • Incubate with primary antibody overnight at 4°C for optimal sensitivity

     • Wash 3-5 times with TBST before and after secondary antibody incubation

  4. Optimization Strategies:

     • If background is high, increase blocking time and washing steps

     • If signal is weak, try longer exposure times or increase antibody concentration

     • Include recombinant BHLH147 protein as a positive control to verify antibody specificity

  These recommendations are based on general practices for plant transcription factor detection and may require further optimization for specific experimental conditions .

• How should researchers prepare plant samples for BHLH147 detection in different tissue types?

  Effective detection of BHLH147 requires tissue-specific sample preparation approaches:

  Tissue Type	Extraction Method	Special Considerations
  Leaves	Liquid nitrogen grinding with RIPA buffer	Young leaves provide better protein yield; collect at similar developmental stages
  Roots	Sonication-assisted extraction with high salt buffer	Wash thoroughly to remove soil particles; include 1% polyvinylpolypyrrolidone (PVPP) to remove phenolics
  Flowers	Liquid nitrogen grinding with nuclear extraction buffer	Separate developmental stages for stage-specific expression analysis
  Seeds	High-pressure homogenization with detergent buffer	Pre-soak seeds for 4-6 hours to soften tissues before extraction
  Cell cultures	Direct lysis in extraction buffer	Harvest cells in logarithmic growth phase for consistent results

  For all tissue types, include protease inhibitors (e.g., 1 mM PMSF, complete protease inhibitor cocktail) and phosphatase inhibitors if studying phosphorylation states. Keep samples at 4°C throughout processing to prevent protein degradation. Consider fractionating samples to enrich for nuclear proteins since BHLH147 is a transcription factor primarily located in the nucleus .

• What are the common challenges in BHLH147 detection and how can they be addressed?

  Researchers frequently encounter several challenges when working with plant transcription factor antibodies like BHLH147:

  1. Low Signal Intensity:

     • Cause: Low abundance of transcription factors or suboptimal extraction

     • Solution: Implement nuclear enrichment protocols; concentrate protein samples using TCA precipitation; increase antibody concentration or extend incubation time

  2. Non-specific Binding:

     • Cause: Cross-reactivity with related bHLH family proteins

     • Solution: Increase blocking time (3-4 hours); use 5% BSA instead of milk for blocking; pre-absorb antibody with plant extracts from bhlh147 knockout lines

  3. Variable Results Between Experiments:

     • Cause: Inconsistent sample preparation or environmental variations affecting plant growth

     • Solution: Standardize growth conditions; harvest tissues at the same time of day; prepare master mixes for buffers and reagents

  4. Signal Interference from Plant Compounds:

     • Cause: Phenolics, polysaccharides, and secondary metabolites interfering with detection

     • Solution: Add PVPP (1-2%) to extraction buffers; include β-mercaptoethanol (2-5 mM); incorporate a TCA/acetone precipitation step

  5. Protein Degradation:

     • Cause: Active proteases in plant tissues

     • Solution: Keep samples cold throughout processing; use multiple protease inhibitors; process samples quickly without delays

  These troubleshooting approaches should be systematically tested when optimizing detection protocols for BHLH147 .

Advanced Research Questions

• How can researchers validate BHLH147 Antibody specificity in knockout/knockdown studies?

  Rigorous validation of antibody specificity is critical for reliable BHLH147 research. A comprehensive validation strategy includes:

  1. Genetic Controls:

     • Generate CRISPR/Cas9 knockout or RNAi knockdown lines targeting BHLH147

     • Compare antibody signal between wild-type and knockout/knockdown lines using Western blotting

     • Signals present in wild-type but absent or significantly reduced in knockout lines confirm specificity

  2. Recombinant Protein Controls:

     • Express recombinant BHLH147 protein with epitope tags (His, GST, or FLAG)

     • Perform side-by-side detection with both anti-tag antibody and BHLH147 antibody

     • Matching band patterns confirm antibody specificity

     • Perform antibody pre-absorption with recombinant protein to eliminate specific signals

  3. Cross-Reactivity Assessment:

     • Express closely related bHLH family proteins (especially BHLH128, which shares sequence similarity)

     • Test antibody against these related proteins to assess potential cross-reactivity

     • Perform sequence alignment analysis to identify unique and conserved epitopes

  4. Mass Spectrometry Validation:

     • Perform immunoprecipitation with BHLH147 antibody

     • Analyze precipitated proteins using LC-MS/MS

     • Confirm presence of BHLH147 peptides in the precipitated fraction

  These validation strategies provide convincing evidence of antibody specificity and should be reported in publications to enhance reproducibility of research findings .

• What approaches can be used to study BHLH147 protein-protein interactions and DNA binding?

  Advanced studies of BHLH147 function require specialized techniques:

  1. Protein-Protein Interaction Studies:

     • Co-Immunoprecipitation (Co-IP): Use BHLH147 antibody to pull down protein complexes from plant nuclear extracts, followed by Western blotting or mass spectrometry to identify interacting partners

     • Yeast Two-Hybrid Screening: Create BHLH147 bait constructs to screen for interacting proteins

     • Bimolecular Fluorescence Complementation (BiFC): Visualize interactions in planta by fusing BHLH147 and potential partners to split fluorescent protein fragments

     • Proximity Labeling: Fuse BHLH147 to BioID or APEX2 for in vivo labeling of proximal proteins

  2. DNA-Binding Studies:

     • Chromatin Immunoprecipitation (ChIP): Use BHLH147 antibody to identify genomic binding sites in vivo

     • Electrophoretic Mobility Shift Assay (EMSA): Assess direct binding to specific DNA sequences in vitro

     • DNA Affinity Purification Sequencing (DAP-seq): Identify genome-wide binding sites using purified protein

     • ChIP-sequencing: Map genome-wide binding sites under different conditions

  3. Dynamic Regulation Studies:

     • Phosphorylation Analysis: Use phospho-specific antibodies or mass spectrometry to identify post-translational modifications

     • Proteasome Inhibition: Assess protein stability using MG132 treatment and BHLH147 antibody detection

     • Nuclear-Cytoplasmic Fractionation: Monitor subcellular localization changes using the antibody on fractionated samples

  These methodologies provide mechanistic insights into how BHLH147 regulates target genes and participates in larger regulatory networks .

• How can researchers utilize the BHLH147 Antibody in high-throughput phenotypic screening approaches?

  The BHLH147 Antibody can be adapted for high-throughput screening using these advanced methodologies:

  1. Automated Immunostaining Platforms:

     • Develop protocols for robotic liquid handling systems to process multiple samples

     • Standardize fixation, permeabilization, and staining conditions for consistent results

     • Implement positive and negative controls in each plate for quality assurance

  2. High-Content Imaging Analysis:

     • Perform immunofluorescence with BHLH147 antibody in multi-well formats

     • Quantify subcellular localization, intensity, and pattern changes across treatment conditions

     • Apply machine learning algorithms to identify subtle phenotypic changes

  3. Reverse Phase Protein Arrays (RPPA):

     • Spot multiple plant extracts on nitrocellulose-coated slides

     • Probe with BHLH147 antibody and fluorescent secondary antibodies

     • Quantify protein levels across hundreds of samples simultaneously

  4. Integration with -omics Data:

     • Correlate BHLH147 protein levels with transcriptomic data across conditions

     • Use antibody-based chromatin profiling in conjunction with RNA-seq for integrated analyses

     • Create regulatory network models based on protein level changes and downstream effects

  5. Mutant/Treatment Screening:

     • Apply across genetic libraries or chemical treatment collections

     • Identify conditions that alter BHLH147 levels or localization

     • Use as a readout for stress responses or developmental transitions

  This approach enables researchers to comprehensively assess BHLH147 function across multiple genetic backgrounds, environmental conditions, and developmental stages in a time-efficient manner .

• What methodologies combine BHLH147 Antibody with advanced imaging techniques for subcellular localization studies?

  Contemporary plant cell biology offers several sophisticated approaches for visualizing BHLH147:

  1. Super-Resolution Microscopy:

     • Stimulated Emission Depletion (STED) Microscopy: Achieves 30-80 nm resolution for precise nuclear localization

     • Stochastic Optical Reconstruction Microscopy (STORM): Single-molecule localization providing 20 nm resolution

     • Structured Illumination Microscopy (SIM): Doubles conventional resolution to ~100 nm

     • Sample Preparation: Use thin sections (5-10 μm) of fixed plant tissues; optimize antibody concentration (typically 1:100 to 1:200) for each technique

  2. Live Cell Imaging Combined with Immunodetection:

     • Correlative Light and Electron Microscopy (CLEM): Combine fluorescence imaging of BHLH147 with ultrastructural context

     • Photoactivatable Fluorescent Protein Tags: Track dynamics before fixation and antibody confirmation

     • Single-Molecule Tracking: Follow individual BHLH147 molecules using Fab fragments from the antibody

  3. Multi-Channel Co-Localization:

     • Combine BHLH147 antibody with markers for nuclear subcompartments (nucleolus, chromatin, nuclear speckles)

     • Use spectral unmixing for clear separation of fluorophores

     • Quantify co-localization using Pearson's or Mander's coefficients

  4. Tissue Clearing Techniques:

     • Apply ClearSee or PEA-CLARITY protocols for whole-mount immunostaining

     • Visualize BHLH147 distribution throughout intact plant organs

     • Use light-sheet microscopy for rapid 3D imaging with minimal photobleaching

  These advanced imaging approaches reveal not just the presence of BHLH147 but its precise subnuclear organization, potentially identifying distinct nuclear bodies or chromatin domains associated with this transcription factor .

• How can researchers apply systems biology approaches using BHLH147 Antibody data?

  Integration of BHLH147 antibody-generated data into systems biology frameworks enables comprehensive understanding of regulatory networks:

  1. Multi-omics Data Integration:

     • Correlate BHLH147 protein levels (determined by quantitative immunoblotting) with transcriptome profiling data

     • Map ChIP-seq binding sites to changes in proteome composition under varying conditions

     • Construct directed protein-DNA interaction networks with BHLH147 as a central node

  2. Mathematical Modeling of Transcriptional Circuits:

     • Use antibody-derived protein concentration data as input parameters for differential equation models

     • Incorporate protein half-life measurements (using cycloheximide chase assays with the antibody) into dynamic simulations

     • Validate model predictions by measuring BHLH147 levels in perturbed systems

  3. Network Perturbation Analysis:

     • Quantify BHLH147 protein levels across multiple genetic backgrounds (mutants, overexpressors)

     • Measure downstream effects on target proteins using multiplex immunoassays

     • Identify conditional dependencies and feedback loops in the regulatory network

  4. Temporal and Spatial Mapping of Regulatory Dynamics:

     • Create time-course profiles of BHLH147 expression across developmental stages

     • Map tissue-specific patterns using immunohistochemistry with the antibody

     • Build 4D digital models integrating temporal and spatial expression data

  Sample data integration framework:

  Data Type	Technique Using BHLH147 Antibody	Integration Approach
  Protein Abundance	Quantitative Western Blot	Direct parameter input for models
  DNA Binding Sites	ChIP-seq	Network edge definition (protein→gene)
  Protein Interactions	Co-IP + MS	Protein complex assembly rules
  Subcellular Localization	Immunofluorescence	Compartment-specific reaction constraints
  Protein Stability	Cycloheximide Chase	Dynamic parameter estimation

  This systems-level analysis reveals emergent properties not discernible from individual experiments and places BHLH147 within the broader context of plant regulatory networks .

• What are the technical considerations for using BHLH147 Antibody with plant samples containing high levels of interfering compounds?

  Plant tissues often contain compounds that interfere with antibody-based detection. Advanced researchers should consider these specialized protocols:

  1. Specialized Extraction Buffers for Problematic Tissues:

     • For phenolic-rich tissues (e.g., stress-treated leaves, seeds):

       • Add 2-5% PVPP or 1-2% PVP-40 to extraction buffer

       • Include 10-50 mM sodium ascorbate and 10 mM DTT as antioxidants

       • Consider extraction in the presence of Amberlite XAD-4 resin to absorb phenolics

     • For tissues with high secondary metabolites:

       • Implement a two-phase extraction with Tris-saturated phenol

       • Follow with methanol/ammonium acetate precipitation to remove contaminants

  2. Sample Clean-up Methods for Immunodetection:

     • TCA/acetone precipitation followed by wash steps with 80% acetone

     • Spin column-based protein purification with specialized plant protein kits

     • Size exclusion chromatography for removal of small molecule inhibitors

     • Immunoprecipitation with protein A/G beads to isolate BHLH147 before analysis

  3. Modified Immunodetection Protocols:

     • Add 0.1-0.5% plant-derived protein blockers (e.g., non-specific plant extract from unrelated species)

     • Increase detergent concentration (0.1-0.3% Tween-20) in wash buffers

     • Use signal enhancement systems (tyramide signal amplification) for low abundance detection

     • Implement lengthy wash protocols (5-7 washes of 10 minutes each) to reduce background

  4. Validation Approaches for Complex Samples:

     • Include recombinant BHLH147 protein spiked into plant extracts as positive controls

     • Run parallel samples with increasing concentrations of potentially interfering compounds

     • Perform epitope competition assays to distinguish specific from non-specific signals

     • Use transgenic plants expressing epitope-tagged BHLH147 as reference standards

  These specialized methodologies are particularly important when studying BHLH147 under stress conditions that induce secondary metabolite production or when working with plant tissues naturally high in interfering compounds such as seeds or woody tissues .