BHLH117 Antibody

What is BHLH117 and what is its functional role in plant biology?

BHLH117 belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors found in Arabidopsis thaliana (Mouse-ear cress). Like other bHLH proteins, it likely binds to DNA via specific E-box motifs (5'-CANNTG-3') to regulate gene expression . While the specific function of BHLH117 is not fully characterized in the provided literature, bHLH transcription factors typically play crucial roles in plant development, stress responses, and metabolic regulation. By comparison with other bHLH family members, these proteins often act as transcriptional activators or repressors through protein-protein interactions with other transcription factors or cofactors.

What are the key specifications of commercial BHLH117 Antibodies?

BHLH117 Antibody is typically generated as a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana BHLH117 protein . The antibody is supplied in liquid form containing preservatives (such as 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . These antibodies undergo purification through antigen affinity methods to ensure specificity. The expected molecular weight of the target protein should be determined based on the amino acid sequence, similar to how other bHLH proteins have predicted versus observed molecular weights (e.g., BHLHA15 with a predicted weight of 21 kDa and observed weight of 24 kDa) .

What validated applications are appropriate for BHLH117 Antibody?

BHLH117 Antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . When designing experiments with this antibody, researchers should consider using positive controls for validation. Similar to approaches with other bHLH antibodies, whole cell lysates containing the target protein can serve as effective positive controls for applications such as ELISAs, immunoprecipitation, and Western blotting . For Western blotting specifically, researchers should follow standard protocols while optimizing antibody dilution. While immunohistochemistry applications are not explicitly validated for BHLH117 Antibody, other bHLH family antibodies have been used successfully in IHC-P applications, suggesting potential for similar applications after proper validation .

How should I optimize Western blot protocols for BHLH117 Antibody?

When optimizing Western blot protocols for BHLH117 Antibody, follow these methodological steps:

• Sample preparation: Extract proteins from Arabidopsis thaliana tissue using standard lysis buffers containing protease inhibitors to prevent degradation.

• Protein separation: Separate proteins using SDS-PAGE with appropriate percentage gels (10-12% recommended for most transcription factors).

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block nonspecific binding sites with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Incubate with BHLH117 Antibody at an initial dilution of 1-2 μg/ml (similar to recommendations for other bHLH antibodies) . Optimize concentration based on signal-to-noise ratio.

• Secondary antibody: Use appropriate anti-rabbit IgG-HRP conjugated secondary antibody.

• Detection: Develop using enhanced chemiluminescence (ECL) substrates.

Include appropriate controls: positive control (tissue known to express BHLH117), negative control (tissue not expressing the target), and loading control (housekeeping protein like GAPDH or actin).

What are the recommended storage conditions for maintaining BHLH117 Antibody activity?

BHLH117 Antibody should be stored at -20°C or -80°C upon receipt to maintain optimal activity . Avoid repeated freeze-thaw cycles as they can degrade antibody performance through protein denaturation and aggregation. For working solutions, store at 4°C for short-term use (up to three months), similar to storage recommendations for other bHLH family antibodies . When preparing working dilutions, use buffers containing stabilizing proteins (e.g., 1% BSA) and consider adding sodium azide (0.02%) for solutions stored more than a week. Always centrifuge briefly before use to collect all solution at the bottom of the tube and remove any potential aggregates.

How can I validate the specificity of BHLH117 Antibody in my experimental system?

Validating antibody specificity is critical for reliable research results. For BHLH117 Antibody, implement these rigorous validation approaches:

• Genetic validation: Use BHLH117 knockout/knockdown plants as negative controls. The absence of signal in these samples confirms specificity.

• Recombinant protein competition: Pre-incubate the antibody with purified BHLH117 recombinant protein before application to samples. Signal reduction indicates specific binding.

• Overexpression systems: Test the antibody in systems overexpressing tagged BHLH117 and confirm colocalization of antibody signal with the tag.

• Multiple antibody comparison: If available, compare results with another antibody targeting a different epitope of BHLH117.

• Mass spectrometry: For definitive validation, perform immunoprecipitation followed by mass spectrometry to confirm the identity of pulled-down proteins.

When analyzing Western blot results, be aware that observed molecular weights may differ from predicted weights due to post-translational modifications, similar to observations with BHLHA15 (predicted: 21 kDa, observed: 24 kDa) .

How do bHLH family proteins compare structurally and functionally, and what implications does this have for antibody research?

bHLH proteins share a conserved basic helix-loop-helix domain but exhibit diverse functions. This structural comparison is important for understanding potential cross-reactivity:

The conserved DNA-binding motifs across bHLH family members indicate similar molecular mechanisms but with tissue-specific and context-dependent functions. When using BHLH117 Antibody, researchers should be aware of potential cross-reactivity with other bHLH family members that share structural homology. Perform additional validation steps if working in systems that express multiple bHLH proteins.

What troubleshooting approaches are recommended for weak or inconsistent signals with BHLH117 Antibody?

When encountering signal issues with BHLH117 Antibody, systematically address potential causes:

• Protein expression levels: BHLH117 may be expressed at low levels or in specific developmental stages or stress conditions. Verify expected expression using qRT-PCR before antibody testing.

• Epitope accessibility: If the epitope is masked by protein interactions or conformational changes, modify lysis conditions (try different detergents or denaturing conditions).

• Antibody concentration: Test a range of concentrations, potentially higher than standard dilutions (similar to recommendations for BHLHA15 starting at 5 μg/mL for IHC-P) .

• Incubation conditions: Extend primary antibody incubation time (overnight at 4°C rather than 1-2 hours at room temperature).

• Detection system: Use more sensitive detection methods, such as enhanced chemiluminescence substrates for Western blotting.

• Protein extraction: For plant tissues, use specialized extraction buffers that effectively solubilize membrane-associated transcription factors.

• Batch variability: If changing lots, perform side-by-side testing with previous successful experiments.

How can BHLH117 Antibody be utilized in studies of plant transcription networks?

BHLH117 Antibody can be instrumental in elucidating plant transcriptional networks through these methodological approaches:

• Chromatin Immunoprecipitation (ChIP): Use BHLH117 Antibody to identify DNA binding sites and target genes. This approach helps map the regulatory network controlled by BHLH117.

• Co-Immunoprecipitation (Co-IP): Identify protein interaction partners of BHLH117, revealing potential transcriptional complexes that regulate gene expression.

• Immunolocalization: Determine the subcellular localization of BHLH117 under different environmental conditions or developmental stages.

• Protein expression analysis: Monitor BHLH117 protein levels in response to various stresses or developmental cues, complementing transcriptomic data.

• Comparative studies: Analyze BHLH117 expression patterns across different plant tissues, developmental stages, or in response to environmental stimuli.

This multi-faceted approach provides insights into how BHLH117 contributes to transcriptional regulation networks in Arabidopsis thaliana, similar to approaches used for studying other transcription factors like those in the E-box binding protein family .

What considerations should be made when designing experiments investigating BHLH117 interactions with other transcription factors?

When investigating BHLH117 interactions with other transcription factors, consider these methodological aspects:

• Co-expression analysis: Before conducting protein interaction studies, verify which potential interaction partners are co-expressed with BHLH117 in the same tissues/conditions.

• Protein complex preservation: Use gentle lysis conditions that preserve native protein-protein interactions (non-ionic detergents like NP-40 or Triton X-100).

• Cross-linking approaches: Consider in vivo cross-linking before lysis to capture transient interactions that might be lost during purification.

• Reciprocal Co-IP: Confirm interactions by performing Co-IP with antibodies against both BHLH117 and the suspected interaction partner.

• Controls for specificity: Include negative controls (unrelated antibodies) and competition assays to ensure specificity of observed interactions.

• Yeast two-hybrid or BiFC validation: Validate direct interactions using orthogonal methods like yeast two-hybrid or bimolecular fluorescence complementation.

These approaches are similar to methods used to study other bHLH protein interactions, such as the interactions between ID3 and E-proteins (E12/E47) that regulate gene expression in other biological systems .

How should researchers interpret multiple bands in Western blots using BHLH117 Antibody?

Multiple bands in Western blots using BHLH117 Antibody may have several biological or technical explanations:

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can alter protein migration. This is consistent with observations in other bHLH proteins, where observed molecular weights may differ from predicted weights (e.g., BHLHA15 with predicted 21 kDa vs. observed 24 kDa) .

• Isoforms: Alternative splicing can generate multiple protein isoforms of different sizes.

• Proteolytic processing: The protein may undergo processing that generates fragments recognized by the antibody.

• Cross-reactivity: The antibody may recognize other bHLH family members with similar epitopes.

To determine which band represents BHLH117:

• Compare band patterns in tissues known to express high versus low levels of BHLH117

• Use BHLH117 knockout/knockdown samples as negative controls

• Compare with tagged recombinant BHLH117 of known size

• Perform peptide competition assays to identify specific versus non-specific bands

What experimental approaches can distinguish between direct and indirect effects of BHLH117 on gene expression?

To distinguish between direct and indirect effects of BHLH117 on gene regulation:

• Chromatin Immunoprecipitation (ChIP): Use BHLH117 Antibody for ChIP followed by sequencing (ChIP-seq) to identify direct binding sites of BHLH117 on genomic DNA. This approach identifies genome-wide binding patterns, similar to methods used to study other bHLH proteins and their binding to E-box motifs .

• Inducible expression systems: Create plant lines with inducible BHLH117 expression and analyze immediate early gene responses (within hours of induction) before secondary effects occur.

• Promoter-reporter assays: Test direct activation/repression of suspected target gene promoters by BHLH117 in transient expression systems, similar to promoter-reporter assays used to study ID3 inhibition of E2A protein activation .

• Motif analysis: Identify enrichment of specific DNA binding motifs (likely E-box variants) in BHLH117-regulated genes.

• Protein-DNA binding assays: Perform electrophoretic mobility shift assays (EMSA) with recombinant BHLH117 and DNA fragments containing putative binding sites.

• Combined approaches: Integrate ChIP-seq data with RNA-seq after BHLH117 perturbation to distinguish direct targets (showing both binding and expression changes) from indirect targets.