BHLH125 Antibody

What is bHLH125 and why is it significant in research?

bHLH125 is a transcription factor belonging to the basic helix-loop-helix (bHLH) family. It functions as a DNA-binding protein that regulates gene expression in various biological processes. The significance of bHLH125 stems from its role in transcriptional regulation processes in both plants and animals . In plants like Chenopodium quinoa (quinoa), it is annotated as "transcription factor bHLH125-like" (LOC110702386) . bHLH transcription factors are involved in numerous biological functions including neurogenesis, myogenesis, hematopoiesis, environmental responses, circadian rhythm regulation, and cell cycle/proliferation control . Research into bHLH125 antibodies allows for deeper investigation of these regulatory mechanisms.

What are the key structural characteristics of bHLH125 that influence antibody design?

The bHLH125 protein contains a conserved basic helix-loop-helix domain consisting of two alpha helices connected by a loop region. The basic region at the N-terminal end of the first helix is rich in basic amino acids and is responsible for DNA contact, while the HLH region is involved in dimerization .

Key structural considerations for antibody design include:

• The basic region contains highly conserved residues, particularly at positions 1, 2, 5, 6, 8, 9, 12, and 13, which make base-specific contacts with DNA

• Residues in positions 1, 2, 4, 6, 8, 10, 12-14, 17, 47-51 interact non-specifically with DNA's phosphate backbone

• bHLH proteins typically form homo- or heterodimers through their HLH domains

• The protein may undergo post-translational modifications that affect its function and antibody recognition

When designing antibodies against bHLH125, researchers must consider the conservation of these domains across bHLH family members to ensure specificity.

How do I select between polyclonal and monoclonal antibodies for bHLH125 research?

The decision between polyclonal and monoclonal antibodies depends on your specific research objectives:

Polyclonal antibodies for bHLH125:

• Advantage: Recognize multiple epitopes, potentially improving detection sensitivity

• Advantage: More tolerant of minor protein denaturation or conformation changes

• Disadvantage: May have higher background and cross-reactivity with related bHLH family members

• Best for: Initial exploratory studies and applications requiring high sensitivity

Monoclonal antibodies for bHLH125:

• Advantage: High specificity for a single epitope

• Advantage: Better lot-to-lot consistency

• Advantage: Can discriminate between closely related bHLH family members if targeted to unique regions

• Disadvantage: May lose reactivity if the target epitope is modified or masked

• Best for: Highly specific applications where distinguishing between closely related bHLH proteins is critical

For studying bHLH125, consider whether the recognition of specific domains (basic region vs. HLH domain) is important for your research question .

What are the essential validation steps for a bHLH125 antibody?

A comprehensive validation strategy for bHLH125 antibodies should include:

• Specificity testing:

  • Western blot analysis showing a single band at the expected molecular weight

  • Comparison with knockout/knockdown samples as negative controls

  • Testing across multiple sample types where bHLH125 expression is expected or not expected

• Sensitivity assessment:

  • Using samples with known varying amounts of bHLH125

  • Detecting endogenous levels in relevant experimental systems

• Application-specific validation:

  • For IHC: testing on fixed tissues with known expression patterns

  • For IP: confirming pull-down of the target protein and known interactors

  • For ChIP: validating by qPCR of known target genes with E-box motifs

• Reproducibility testing:

  • Testing across different lots

  • Independent validation by different researchers

As noted in the literature, "49% of internally generated antibodies failed validation," highlighting the importance of thorough validation before conducting significant research .

How can I distinguish between bHLH125 and other closely related bHLH family members?

Distinguishing between bHLH125 and other bHLH family members requires careful experimental design:

• Epitope selection strategy:

  • Target regions outside the highly conserved basic and HLH domains

  • Focus on unique regions in the N- or C-terminal domains of bHLH125

  • Consider using peptide arrays to identify unique epitopes

• Validation experiments:

  • Perform Western blots on recombinant protein panels of multiple bHLH family members

  • Test antibody reactivity on cell lines with knockouts of specific bHLH proteins

  • Use immunoprecipitation followed by mass spectrometry to confirm specificity

• Cross-reactivity testing:

  • Based on sequence similarity analysis, identify the most closely related bHLHs

  • Test antibody against these related proteins in parallel

  • Consider the amino acid variations at positions 5, 6, 8, 9, and 13 in the basic domain that help classify different bHLH groups

Use this information to predict potential cross-reactivity and design appropriate controls for your experiments .

What are the optimal conditions for using bHLH125 antibodies in Western blotting?

For optimal Western blotting with bHLH125 antibodies:

• Sample preparation:

  • Use fresh samples when possible

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylated forms

  • Use both reducing and non-reducing conditions to account for potential disulfide bonds

• Gel electrophoresis considerations:

  • Use 10-12% acrylamide gels for optimal resolution

  • Include positive controls (recombinant bHLH125) and negative controls

  • Consider native PAGE if conformation is important for antibody recognition

• Transfer and detection optimization:

  • PVDF membranes generally work well for transcription factors

  • Block with 5% non-fat milk or BSA (the latter preferred if detecting phosphorylated forms)

  • Optimize primary antibody concentration (typically 1:500 to 1:2000 dilution)

  • Include longer exposure times to detect low abundance transcription factors

  • Consider signal amplification systems for improved sensitivity

• Validation controls:

  • Run full-length blots to identify potential off-target bands

  • Include molecular weight markers

  • Consider using knockout/knockdown samples as negative controls

When interpreting results, be aware that bHLH transcription factors may show different banding patterns due to post-translational modifications or alternative splicing.

How should I optimize ChIP protocols when using bHLH125 antibodies?

For chromatin immunoprecipitation (ChIP) with bHLH125 antibodies:

• Crosslinking optimization:

  • Standard 1% formaldehyde for 10 minutes works for most transcription factors

  • Consider dual crosslinking with both formaldehyde and protein-specific crosslinkers

  • Quench thoroughly with glycine to prevent over-crosslinking

• Sonication parameters:

  • Aim for 200-500 bp fragments for optimal resolution

  • Verify sonication efficiency by running a small aliquot on an agarose gel

  • Sonication conditions must be optimized for each cell/tissue type

• Immunoprecipitation considerations:

  • Pre-clear lysates to reduce background

  • Determine optimal antibody amount empirically (typically 2-5 μg per reaction)

  • Include IgG controls and input samples

  • Consider using protein A/G beads for most mammalian antibodies

• Target verification:

  • Design primers for qPCR targeting known E-box elements (CANNTG motifs)

  • Include primers for regions without E-box elements as negative controls

  • Consider the specific half-site preferences (CAC, CAT, or CAG) based on bHLH125 subclass

• Data interpretation:

  • Compare enrichment to input DNA (typically expressed as percent input)

  • Analyze binding to canonical E-box motifs (CANNTG) and their flanking sequences

  • Be aware that bHLH factors bind as dimers, with each monomer recognizing a "CAN" half-site

What are the best approaches for using bHLH125 antibodies in co-immunoprecipitation studies?

For effective co-immunoprecipitation (co-IP) studies with bHLH125 antibodies:

• Lysis buffer optimization:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors

  • Consider detergent strength (CHAPS or NP-40 are often gentler than SDS)

  • Test different salt concentrations to balance specificity and maintenance of interactions

• Experimental design considerations:

  • Perform reciprocal IPs when possible (IP with antibodies against both bHLH125 and suspected interactors)

  • For dimerization studies, consider that bHLH proteins form homo- and heterodimers

  • Target likely interactors based on biological function (e.g., other transcription factors like MYB or BZR1 in plant systems)

• Controls:

  • Include IgG control

  • Include lysate-only controls

  • Consider using bHLH125 knockout/knockdown samples

  • Validate specificity with Western blotting before co-IP

• Result analysis:

  • When studying bHLH125 dimerization, be aware that different dimers may have different DNA binding specificities

  • For plant bHLH125, consider interactions with MYB proteins in regulating processes like flavonoid biosynthesis

  • Distinguish between direct and indirect interactions through additional experiments

How do I troubleshoot weak or absent signals when using bHLH125 antibodies?

When facing weak or absent signals with bHLH125 antibodies:

• Expression level verification:

  • Confirm bHLH125 expression in your sample through qRT-PCR

  • Use positive control samples known to express bHLH125

  • Be aware that transcription factors often have low endogenous expression

• Sample preparation issues:

  • Ensure sample is fresh and properly stored

  • Verify protein extraction efficiency with total protein stains

  • Consider nuclear extraction for enrichment of nuclear transcription factors

  • Check for protease activity during sample preparation

• Technical adjustments:

  • Increase antibody concentration

  • Extend incubation time and optimize temperature

  • Use more sensitive detection systems

  • Reduce washing stringency

  • For Western blots, try different membrane types

  • For IHC, optimize antigen retrieval methods

• Antibody-specific concerns:

  • Test a different lot or a different antibody targeting another epitope

  • Verify antibody storage conditions and expiration

  • Consider that the epitope might be masked by protein interactions or modifications

  • For fixed samples, ensure fixation hasn't destroyed the epitope

When troubleshooting, methodically change one variable at a time and document all optimization steps.

How should I interpret contradictory results between different bHLH125 antibodies?

When facing contradictory results between different bHLH125 antibodies:

Remember that "if an antibody detects a protein with an unexpected molecular weight, look for controls that validate that the protein detected is actually the target protein" .

What considerations are important when studying post-translational modifications of bHLH125?

When investigating post-translational modifications (PTMs) of bHLH125:

• Antibody selection strategy:

  • Use modification-specific antibodies (e.g., phospho-specific)

  • Ensure the base antibody recognizes bHLH125 regardless of modification state

  • Validate specificity with appropriate controls (e.g., phosphatase treatment)

• Sample preparation considerations:

  • Include appropriate inhibitors (phosphatase, deacetylase, etc.)

  • Consider enrichment strategies for modified proteins

  • Use gentle lysis conditions to preserve modifications

• Experimental design:

  • Include conditions that would alter modification status (e.g., stimulation, stress)

  • For plant bHLH125, consider treatments that affect response to environmental stresses

  • Use both total and modification-specific antibodies in parallel

  • Consider 2D gel electrophoresis to separate modified forms

• Interpretation framework:

  • PTMs can affect protein-protein interactions, particularly dimerization with other bHLH factors

  • PTMs may alter DNA binding specificity or affinity for E-box motifs

  • Consider that "the activity of many TFs is further modified by dynamic post-translational modifications such as phosphorylation, which can affect their subcellular localization and dimerizing partners"

  • In plant systems, bHLH125 phosphorylation might be involved in stress response pathways

How do I design experiments to study bHLH125 dimerization with other transcription factors?

To study bHLH125 dimerization:

• Partner identification strategies:

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID, APEX)

  • Bioinformatic prediction based on known bHLH dimerization patterns

• Dimerization analysis methods:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation

• Functional analysis of dimers:

  • Luciferase reporter assays with E-box-containing promoters

  • ChIP-seq to identify genomic binding sites of different dimers

  • Mutational analysis of dimerization interfaces

  • Consider DNA binding specificity differences between homodimers and heterodimers

• Data interpretation framework:

  • Different dimer combinations may recognize different E-box variants or half-sites

  • "Each monomer of this dimeric structure contacts half of the E-box CANNTG sequence, but they do it in opposing strands, resulting in each monomer recognizing a 'CAN' half site"

  • Consider that different dimers may have different biological functions

What are the key considerations for using bHLH125 antibodies in different model organisms?

When using bHLH125 antibodies across different model organisms:

• Cross-reactivity assessment:

  • Perform sequence alignment of bHLH125 across species of interest

  • Target conserved epitopes for cross-species reactivity

  • Validate with positive controls from each species

  • Be aware that even with high sequence homology, antibody binding may vary

• Model-specific considerations:

  • Plants (e.g., Arabidopsis, quinoa): Focus on nuclear extraction protocols; consider tissue-specific expression patterns and developmental stages; be aware of the roles in stress responses and flavonoid biosynthesis

  • Mammals: Consider potential interspecies differences in expression patterns and regulation; optimize nuclear extraction protocols

  • Cell lines: Verify endogenous expression levels; consider using overexpression systems for low-abundance targets

• Application optimization by model:

  • For plant tissues: Optimize fixation and antigen retrieval for IHC/IF

  • For animal tissues: Consider tissue-specific background issues

  • For immunoprecipitation: Adjust lysis conditions for different tissue types

• Evolutionary context:

  • Consider that "the phylogenetic tree of bHLH factors inferred from the bHLH domain was found largely aligned with three previously determined groups (A, B, C) based on similarity in binding affinities"

  • This evolutionary relationship can help predict antibody cross-reactivity and function

How can I use bHLH125 antibodies to study its role in stress responses in plants?

For studying bHLH125's role in plant stress responses:

• Experimental design considerations:

  • Apply relevant stresses (salt, drought, temperature, pathogen) with appropriate controls

  • Include time course analysis to capture dynamic responses

  • Consider tissue-specific responses and select appropriate sampling methods

  • Design experiments that distinguish between different stress types

• Analytical approaches:

  • ChIP-seq to identify stress-responsive target genes

  • Co-IP to identify stress-specific interaction partners

  • Immunolocalization to track potential subcellular relocalization during stress

  • Phospho-specific antibodies to detect stress-induced modifications

• Functional validation:

  • Compare wildtype and bHLH125 mutant plants under stress conditions

  • Use reporter constructs to monitor bHLH125 activity during stress

  • Integrate with transcriptomic data to build regulatory networks

  • Consider that bHLH transcription factors can regulate flavonoid synthesis, which may play a role in stress responses

• Data interpretation framework:

  • In Arabidopsis, "the bHLH Transcription Factor HBI1 Mediates the Trade-Off between Growth and Pathogen-Associated Molecular Pattern–Triggered Immunity"

  • "Salt stress activated AtMYC2 through a mitogen-activated protein kinase (MAPK) cascade. Then, the AtMYC2 could bind to the promotor of rate-limiting enzyme P5CS1 in the biosynthesis of proline, thereby regulating the biosynthesis of proline, and thus regulating salt tolerance"

  • Consider that bHLH125 may interact with other transcription factor families (like MYB) in stress response pathways