BHLH101 Antibody

What is BHLH101 and why is it important in plant research?

BHLH101 is a basic helix-loop-helix transcription factor belonging to the bHLH Ib subgroup that includes bHLH38, bHLH39, and bHLH100. These transcription factors play crucial roles in regulating iron homeostasis in plants, particularly Arabidopsis thaliana. Unlike FIT, which is root-specific, bHLH101 and other bHLH Ib genes are ubiquitously expressed throughout the plant .

The importance of BHLH101 is highlighted by the severe phenotypes observed in mutant studies. When all four bHLH Ib transcription factors are knocked out (in a quadruple mutant called bhlh4x), plants display chlorotic leaves, reduced chlorophyll concentration, and significantly impaired growth - phenotypes that resemble the fit mutant and can be rescued by extra iron application . These findings demonstrate that BHLH101, together with other bHLH Ib members, is essential for proper iron uptake and plant survival.

What are the key characteristics of an effective BHLH101 antibody?

An effective BHLH101 antibody should demonstrate several key characteristics for reliable research applications:

• High specificity: The antibody should recognize BHLH101 without cross-reactivity to other closely related bHLH transcription factors, particularly other members of the bHLH Ib subgroup (bHLH38, bHLH39, and bHLH100) which share sequence similarities .

• Appropriate epitope targeting: Ideally, the antibody should be raised against regions of BHLH101 that are accessible in both native and denatured conditions, while avoiding regions involved in protein-protein interactions that might be masked when BHLH101 forms complexes with FIT or other proteins .

• Validated applications: The antibody should be validated for intended applications such as Western blotting, immunoprecipitation, ChIP (Chromatin Immunoprecipitation), or immunolocalization studies.

• Species specificity: Given that BHLH101 research is conducted in different plant models, the antibody should be characterized for cross-reactivity with BHLH101 orthologs in research-relevant species.

When selecting a BHLH101 antibody, researchers should review validation data that demonstrates the antibody can distinguish between BHLH101 and other bHLH family members, especially within the context of their specific experimental system.

How can I verify the specificity of a BHLH101 antibody?

Verifying the specificity of a BHLH101 antibody is critical for research integrity. Follow these methodological approaches:

• Use of genetic controls: Test the antibody in wild-type versus bhlh101 single mutant or bhlh4x quadruple mutant (bhlh38 bhlh39 bhlh100 bhlh101) plant materials. A specific antibody should show signal in wild-type samples but no signal (or significantly reduced signal) in mutant samples .

• Recombinant protein validation: Compare the detection of recombinant BHLH101 protein against other recombinant bHLH family proteins, particularly bHLH38, bHLH39, and bHLH100, which share sequence similarities.

• Immunoprecipitation with mass spectrometry: Perform immunoprecipitation followed by mass spectrometry analysis to confirm that the antibody predominantly pulls down BHLH101 rather than other proteins. This approach has been successfully used in similar studies with bHLH transcription factors, as demonstrated in research with ILR3 (bHLH105) .

• Pre-absorption control: Pre-incubate the antibody with recombinant BHLH101 protein before use in your experiment. If the antibody is specific, this pre-absorption should abolish or significantly reduce signal.

• Multiple antibody comparison: When possible, compare results using different antibodies raised against different epitopes of BHLH101 to confirm consistency of detection patterns.

Maintaining appropriate controls is essential when using BHLH101 antibodies, especially when studying proteins from the same family with high sequence homology.

What are the optimal conditions for using BHLH101 antibody in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is a valuable technique for studying BHLH101 interactions with other proteins. Based on successful approaches used in similar research, follow these methodological guidelines:

• Sample preparation: Extract nuclear proteins from plant tissue (preferably roots) under native conditions using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail, and 1 mM PMSF. For studying interactions dependent on iron status, compare samples from plants grown under iron-sufficient and iron-deficient conditions .

• Cross-linking (optional): For transient or weak interactions, consider using a mild crosslinking agent such as disuccinimidyl suberate (DSS) or formaldehyde (0.1-0.5%) for 10-15 minutes before protein extraction.

• Pre-clearing: Pre-clear the protein extract with protein A/G beads without antibody to reduce non-specific binding.

• Immunoprecipitation: Incubate the pre-cleared extract with BHLH101 antibody overnight at 4°C, followed by addition of protein A/G beads for 2-4 hours. Based on successful Co-IP experiments with similar proteins, use 2-5 μg of antibody per 500 μg of total protein .

• Washing stringency: Use progressive washing with increasing stringency to reduce background while maintaining specific interactions. Begin with 3 washes using IP buffer, followed by 2 washes with higher salt concentration (300 mM NaCl).

• Elution and detection: Elute bound proteins and analyze by immunoblotting with antibodies against suspected interaction partners such as FIT, bHLH38, bHLH39, or bHLH100 .

When investigating BHLH101 interactions, it's important to note that research has demonstrated BHLH101 forms complexes with FIT for functionality, with BHLH101 providing DNA-binding capability while FIT contributes transcriptional activation function .

How should I optimize Western blot protocols for BHLH101 detection?

Optimizing Western blot protocols for BHLH101 detection requires attention to several key parameters:

• Sample preparation: Extract nuclear proteins from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% SDS, 5 mM EDTA, and protease inhibitors. Include phosphatase inhibitors if phosphorylation status is relevant.

• Gel percentage selection: Use 10-12% SDS-PAGE gels for optimal resolution of BHLH101, which has a molecular weight in the range of approximately 30-35 kDa.

• Transfer conditions: For complete transfer of BHLH101 protein, use a wet transfer system with 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3 buffer at 100V for 1 hour or 30V overnight at 4°C.

• Blocking optimization: Test both 5% non-fat dry milk and 3-5% BSA in TBS-T as blocking agents to determine which provides the best signal-to-noise ratio for your specific BHLH101 antibody.

• Antibody dilution and incubation: Start with a 1:1000 dilution of primary antibody and incubate overnight at 4°C. For secondary antibody, use a 1:5000-1:10000 dilution with 1-2 hour incubation at room temperature.

• Enhanced chemiluminescence detection: Use a high-sensitivity ECL substrate for detection, as BHLH101 expression can vary widely depending on iron status conditions.

• Controls: Always include positive controls (tissues with known BHLH101 expression), negative controls (bhlh101 mutant tissues), and loading controls (such as histone H3 for nuclear proteins).

When comparing BHLH101
protein levels between iron-sufficient and iron-deficient conditions, be aware that expression is typically induced under iron deficiency .

What is the recommended protocol for ChIP experiments using BHLH101 antibody?

Chromatin Immunoprecipitation (ChIP) using BHLH101 antibody requires careful optimization to study BHLH101 binding to target gene promoters. Based on research showing that bHLH Ib transcription factors have DNA binding ability , follow these methodological recommendations:

• Tissue selection and crosslinking: Harvest 1-2 grams of fresh plant tissue (preferably roots where iron uptake genes are expressed). Crosslink with 1% formaldehyde for 10 minutes under vacuum, followed by quenching with 125 mM glycine.

• Chromatin preparation: Extract nuclei and sonicate chromatin to achieve fragments of 200-500 bp. Verify fragmentation by agarose gel electrophoresis.

• Immunoprecipitation: Pre-clear chromatin with protein A/G beads, then incubate with BHLH101 antibody overnight at 4°C (use 3-5 μg antibody per ChIP reaction). Include a no-antibody control and, if available, an IgG control.

• Washing and elution: Perform sequential washes with increasing stringency, followed by elution of DNA-protein complexes from beads.

• Reverse crosslinking and DNA purification: Reverse crosslinks by heating samples at 65°C overnight, treat with RNase A and Proteinase K, then purify DNA using column-based methods.

• qPCR analysis: Design primers targeting E-box motifs in promoters of known BHLH101 target genes, particularly IRT1 (IRON-REGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2), which contain binding sites for bHLH Ib transcription factors .

Note: Research has shown that bHLH Ib transcription factors like BHLH101 can bind to the promoter of IRT1 in EMSA (Electrophoretic Mobility Shift Assays) , making this gene a good target for ChIP validation.

How can I use BHLH101 antibody to investigate protein degradation mechanisms?

Investigating BHLH101 protein degradation mechanisms is important for understanding iron homeostasis regulation. Based on research showing that related bHLH transcription factors (bHLH105/ILR3 and bHLH115) undergo ubiquitination and degradation by BRUTUS (BTS) , follow these methodological approaches:

• Degradation kinetics analysis:

  • Treat plant seedlings with the protein synthesis inhibitor cycloheximide (100-200 μM)

  • Harvest tissues at different time points (0, 1, 3, 6, 12 hours)

  • Extract proteins and perform Western blot with BHLH101 antibody to determine protein half-life

  • Compare degradation kinetics in wild-type versus bts mutant backgrounds to assess BTS involvement

• Ubiquitination detection:

  • Treat plants with proteasome inhibitor MG132 (50 μM, 6 hours) to allow accumulation of ubiquitinated proteins

  • Immunoprecipitate BHLH101 using its specific antibody

  • Perform Western blot with anti-ubiquitin antibody to detect ubiquitinated forms of BHLH101

  • Include wild-type and bts mutant samples for comparison

• Interaction domain mapping:

  • Create truncated versions of BHLH101 protein lacking potential degradation motifs

  • Express these constructs in plants or protoplasts

  • Use co-immunoprecipitation with BTS to identify interaction domains

  • Similar to the approach used for bHLH105, analyze the C-terminal region for potential BTS-interaction domains (BID)

• Phosphorylation status assessment:

  • Use phosphatase treatment of protein extracts before Western blotting

  • Compare migration patterns to determine if phosphorylation affects BHLH101 stability

  • Use phospho-specific antibodies if available

This methodological approach will help determine whether BHLH101, like related bHLH transcription factors, contains a BTS-interaction domain (BID) and undergoes similar regulatory degradation in response to iron status .

How can I differentiate between BHLH101 and other bHLH Ib transcription factors in my experiments?

Differentiating between BHLH101 and other bHLH Ib transcription factors (bHLH38, bHLH39, and bHLH100) is challenging due to their sequence similarity and functional redundancy. Implement these methodological strategies:

• Epitope selection for antibody generation:

  • Target unique regions of BHLH101 that differ from other bHLH Ib proteins

  • Consider raising antibodies against the most divergent regions, typically found outside the conserved bHLH domain

  • Validate antibody specificity against recombinant versions of all four bHLH Ib proteins

• Genetic approaches:

  • Use single mutants (bhlh101), double/triple mutants, and the quadruple mutant (bhlh38 bhlh39 bhlh100 bhlh101) for validation

  • Complement with transgenic lines expressing tagged versions of each bHLH protein

  • Design gene-specific primers for RT-qPCR to distinguish expression patterns

• Mass spectrometry-based identification:

  • Use immunoprecipitation followed by mass spectrometry

  • Analyze peptide sequences unique to BHLH101 versus other bHLH Ib proteins

  • Quantify relative abundance of each bHLH protein in different tissues and conditions

• Subcellular localization patterns:

  • Leverage different localization patterns of bHLH Ib proteins (BHLH100 and BHLH101 are primarily nuclear, while BHLH38 and BHLH39 show cytoplasmic localization without FIT)

  • Use immunofluorescence with BHLH101 antibody combined with subcellular markers

This comprehensive approach will help distinguish BHLH101 from other functionally redundant family members in experimental contexts.

What are the best methods to study BHLH101-FIT complex formation using antibodies?

Studying BHLH101-FIT complex formation is crucial for understanding iron homeostasis regulation, as research has demonstrated these proteins form a functional transcription complex where BHLH101 provides DNA binding ability while FIT contributes transcriptional activation . Implement these methodological approaches:

• Sequential ChIP (ChIP-reChIP):

  • Perform first ChIP with BHLH101 antibody

  • Elute complexes under mild conditions

  • Perform second ChIP with FIT antibody

  • This confirms co-occupancy of both proteins at the same DNA regions

• Proximity Ligation Assay (PLA):

  • Use primary antibodies against BHLH101 and FIT

  • Apply species-specific secondary antibodies with attached oligonucleotides

  • If proteins are in close proximity, oligonucleotides hybridize and can be amplified

  • Visualize with fluorescence microscopy to detect and quantify interactions in situ

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Similar to approaches used for bHLH121 interaction studies

  • Express BHLH101 fused to C-terminal part of YFP

  • Express FIT fused to N-terminal part of YFP

  • Co-transform into plant cells and observe reconstituted fluorescence

  • Use BHLH101 antibody to confirm expression levels of the fusion protein

• Co-immunoprecipitation with size exclusion chromatography:

  • Perform co-IP with BHLH101 antibody

  • Fractionate eluted complexes by size exclusion chromatography

  • Analyze fractions by immunoblotting with both BHLH101 and FIT antibodies

  • Identify the size of native protein complexes

These approaches provide complementary information about BHLH101-FIT complex formation, from in vitro biochemical evidence to in vivo cellular localization of the interaction.

How can I troubleshoot non-specific binding issues with BHLH101 antibody?

Non-specific binding is a common challenge when working with antibodies against transcription factors like BHLH101. Follow these methodological troubleshooting steps:

• Optimize blocking conditions:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic non-specific interactions

• Adjust antibody concentration and incubation conditions:

  • Perform a dilution series (1:500 to 1:5000) to find optimal concentration

  • Compare overnight incubation at 4°C versus shorter incubations at room temperature

  • Add 0.1-0.2% BSA to antibody dilution buffer to reduce non-specific binding

• Increase washing stringency:

  • Extend washing times (5-10 minutes per wash)

  • Increase the number of washes (5-6 times)

  • Adjust salt concentration in wash buffers (150-500 mM NaCl)

• Pre-absorb the antibody:

  • Incubate diluted antibody with protein extract from bhlh101 mutant or bhlh4x quadruple mutant plants

  • Remove non-specific antibodies by centrifugation before using in your experiment

• Competition assay:

  • Pre-incubate antibody with excess recombinant BHLH101 protein

  • Compare results with and without competition to identify specific bands

If non-specific binding persists, consider generating new antibodies against unique epitopes of BHLH101 or using tagged versions of the protein in transgenic plants.

What controls should I include when studying BHLH101 expression under iron deficiency conditions?

When studying BHLH101 expression under iron deficiency conditions, proper controls are essential for reliable data interpretation. Implement these methodological controls:

• Genetic controls:

  • Wild-type plants (positive control)

  • bhlh101 single mutant (negative control for BHLH101 protein)

  • fit mutant (regulatory network control, as FIT affects BHLH101 expression)

  • bhlh4x quadruple mutant (complete pathway control)

• Treatment controls:

  • Iron-sufficient growth conditions (+Fe, typically 50-100 μM Fe-EDTA)

  • Iron-deficient conditions (-Fe, typically using iron chelators)

  • Time course samples (6h, 12h, 24h, 48h, 72h after transfer to -Fe)

  • Recovery samples (plants returned to +Fe after period of deficiency)

• Antibody controls:

  • Primary antibody omission control

  • Non-specific IgG control

  • Peptide competition assay (pre-incubation with immunizing peptide)

• Marker gene expression:

  • Analyze IRT1 and FRO2 expression as established markers of iron deficiency response

  • These genes should show increased expression under -Fe conditions

• Technical controls for Western blotting:

  • Loading controls (nuclear protein: histone H3; cytoplasmic protein: actin)

  • Standard curve with recombinant BHLH101 for quantification

  • Membrane staining (Ponceau S) to verify equal protein loading

These controls will help distinguish between specific regulation of BHLH101 and general effects of iron deficiency stress on plant physiology.

How can I quantitatively analyze BHLH101 protein levels in relation to transcriptional activity?

Establishing connections between BHLH101 protein levels and transcriptional activity requires integrated analysis of multiple parameters. Implement these methodological approaches:

• Parallel protein and mRNA analysis:

  • Extract protein and RNA from the same tissue samples

  • Quantify BHLH101 protein by Western blot with BHLH101 antibody

  • Measure BHLH101 mRNA levels by RT-qPCR

  • Calculate protein/mRNA ratios to assess post-transcriptional regulation

• Target gene expression correlation:

  • Measure expression of established BHLH101 target genes (IRT1, FRO2)

  • Plot target gene expression against BHLH101 protein levels

  • Calculate Pearson or Spearman correlation coefficients

  • Create a mathematical model of the relationship

• ChIP-qPCR quantification:

  • Perform ChIP with BHLH101 antibody

  • Quantify binding to target promoters by qPCR

  • Normalize to input DNA and IgG control

  • Create binding profiles across multiple target genes

• Integrated multi-omics analysis:

  • Combine proteomics, transcriptomics, and ChIP-seq data

  • Use principle component analysis to identify patterns

  • Apply machine learning algorithms to predict transcriptional outcomes based on BHLH101 protein levels

This integrated approach will provide insights into how BHLH101 protein levels correlate with its DNA-binding activity and target gene expression, advancing understanding of iron homeostasis regulation.

How does the interaction between BHLH101 and BTS affect iron homeostasis regulation?

The interaction between BHLH101 and BRUTUS (BTS) represents an important regulatory mechanism in iron homeostasis. While direct evidence for BHLH101-BTS interaction is limited in the search results, research on related bHLH transcription factors provides a methodological framework to investigate this interaction:

• Protein stability analysis:

  • Compare BHLH101 protein levels in wild-type versus bts mutant plants using BHLH101 antibody

  • Determine if BTS affects BHLH101 stability similar to its effect on bHLH105 and bHLH115, which are both ubiquitinated and degraded by BTS

  • Analyze protein half-life using cycloheximide chase assays in different genetic backgrounds

• BTS-interaction domain (BID) identification:

  • Based on findings that bHLH105 and bHLH115 contain BIDs in their C-terminal regions

  • Generate truncated versions of BHLH101 similar to those created for bHLH105

  • Focus on C-terminal regions, particularly residues similar to the PVA sequence found in bHLH105 and bHLH115

  • Test interaction using yeast two-hybrid and co-immunoprecipitation with BHLH101 antibody

• Iron-dependent regulation analysis:

  • Investigate if the BHLH101-BTS interaction is modulated by iron availability

  • Compare interaction strength under iron-sufficient versus iron-deficient conditions

  • Determine if iron binding to BTS affects its ability to interact with and degrade BHLH101

• Ubiquitination site mapping:

  • Identify potential ubiquitination sites on BHLH101

  • Generate lysine-to-arginine mutants to prevent ubiquitination

  • Test if these mutations affect BTS-mediated degradation

Understanding the BHLH101-BTS interaction would provide insights into the negative regulatory mechanisms that prevent excessive iron uptake, complementing our knowledge of the positive regulatory role of BHLH101 in iron acquisition .

What are the latest advancements in understanding the BHLH101-FIT transcriptional complex?

Recent research has revealed important insights into the BHLH101-FIT transcriptional complex and its role in iron homeostasis. Based on the search results, here are the key methodological advancements:

• Functional complementarity in the transcriptional complex:

  • Recent studies have demonstrated that bHLH Ib transcription factors (including BHLH101) and FIT form a functional complex with complementary roles

  • BHLH101 possesses DNA binding ability but lacks transcriptional activation capacity

  • FIT has transcriptional activation ability but lacks DNA binding capability

  • Together, they form a complete transcriptional complex where BHLH101 targets specific DNA sequences and FIT activates transcription

• DNA binding specificity analysis:

  • Electrophoretic mobility shift assays (EMSA) have shown that BHLH101 and other bHLH Ib proteins can bind to the promoter of IRT1, while FIT cannot

  • The bHLH domain of BHLH101 contains the conserved H-E-R motif at positions 5, 9, and 13, which is critical for DNA binding

  • FIT has a T-E-R motif, explaining its lack of DNA binding ability

• Subcellular localization patterns:

  • Fluorescence microscopy studies have revealed that BHLH101 is primarily localized in the nucleus, even in the absence of FIT

  • In contrast, BHLH38 and BHLH39 require FIT for nuclear accumulation

  • This differential localization may explain functional differences between bHLH Ib members

• Interdependent regulation:

  • Genetic analyses have established that FIT and bHLH Ib proteins (including BHLH101) depend on each other to regulate iron deficiency responses

  • The quadruple bhlh4x mutant (bhlh38 bhlh39 bhlh100 bhlh101) phenocopies the fit mutant

  • This indicates that despite their functional complementarity, neither component can function effectively without the other

These findings provide a mechanistic explanation for why FIT and BHLH101 interdependently regulate iron uptake, advancing our understanding of transcriptional regulation in plant iron homeostasis .

How can BHLH101 antibodies be used to study transcriptional regulatory networks beyond iron homeostasis?

BHLH101 antibodies can be valuable tools for investigating broader transcriptional networks beyond iron homeostasis. Implement these methodological approaches to expand research horizons:

• ChIP-seq for global binding site identification:

  • Perform chromatin immunoprecipitation with BHLH101 antibody followed by next-generation sequencing

  • Analyze under multiple stress conditions (iron deficiency, drought, salt, pathogen infection)

  • Identify novel BHLH101 target genes beyond the known iron uptake pathway

  • Look for enriched motifs in binding regions to refine understanding of BHLH101 DNA binding specificity

• Interactome analysis:

  • Use BHLH101 antibody for immunoprecipitation followed by mass spectrometry

  • Compare protein interaction networks under different environmental conditions

  • Identify novel interaction partners beyond the known iron-related transcription factors

  • Investigate connections to other stress response pathways

• Integration with hormone signaling pathways:

  • Study BHLH101 protein levels and localization in response to different plant hormones

  • Investigate potential crosstalk with ethylene, jasmonate, and auxin signaling pathways

  • Determine if BHLH101 participates in hormone-regulated developmental processes

• Tissue-specific regulatory networks:

  • Use BHLH101 antibody for immunohistochemistry to map tissue-specific expression patterns

  • Compare with known developmental regulators

  • Investigate potential roles in tissue differentiation and organ development

  • Create tissue-specific interactome maps

This expanded research focus could reveal unexpected roles for BHLH101 in coordinating iron homeostasis with broader plant developmental and stress response programs, potentially identifying it as a hub protein connecting multiple regulatory networks.