BHLH87 Antibody

What is bHLH87 and why is it important in plant research?

bHLH87 is a basic helix-loop-helix transcription factor belonging to the bHLH VIII subfamily . It plays critical roles in plant defense mechanisms and hormone signaling pathways. The protein has been identified in various plant species including Arabidopsis thaliana (AtbHLH87), jujube (ZjbHLH87), and pumpkin (CmbHLH87) . bHLH87 is of particular interest because it interacts with pathogen effectors and influences plant resistance to diseases such as powdery mildew. For instance, in pumpkin, CmbHLH87 improves resistance to powdery mildew caused by Podosphaera xanthii . Additionally, it functions in hormone signaling pathways, particularly as a negative regulator of gibberellin (GA) signaling .

How is bHLH87 structurally characterized?

bHLH87 contains the characteristic basic helix-loop-helix domain that defines this transcription factor family. The protein is relatively acidic with a pI of approximately 5.96 and a molecular weight of about 39.1 kDa, as demonstrated in pumpkin CmbHLH87 . Structural analysis using phylogenetic approaches confirms its classification within the bHLH VIII subfamily . The protein predominantly localizes to the nucleus, consistent with its function as a transcription factor . Different plant species show high conservation of the bHLH domain while exhibiting some variation in other regions of the protein.

What criteria should be considered when selecting a bHLH87 antibody for plant research?

When selecting a bHLH87 antibody, researchers should consider:

• Species specificity: Ensure the antibody recognizes bHLH87 from your species of interest. While there is significant homology between bHLH87 proteins across plant species (such as AtbHLH87 and ZjbHLH87), species-specific variations exist .

• Domain recognition: Determine whether the antibody recognizes the conserved bHLH domain or species-specific regions. For cross-species applications, antibodies targeting the conserved bHLH domain may be more versatile.

• Application compatibility: Validate that the antibody works in your intended applications (Western blot, immunoprecipitation, immunofluorescence).

• Validation data: Look for evidence of antibody specificity, such as single band detection in Western blots at the expected molecular weight (~39 kDa for CmbHLH87) .

• Cross-reactivity: Check for potential cross-reactivity with other bHLH family members, which is particularly important given the large size of the bHLH family (171 members in Arabidopsis) .

How can I validate a bHLH87 antibody for research applications?

A comprehensive validation strategy should include:

• Western blot analysis: Using plant tissues with known bHLH87 expression levels, confirm single band detection at the expected molecular weight (~39 kDa) .

• Positive and negative controls: Include samples from:

  • Wild-type plants

  • bHLH87 overexpression lines (such as those described in the pumpkin CmbHLH87 study)

  • bHLH87 knockout/knockdown lines (if available)

• Immunoprecipitation validation: Perform Co-IP experiments with tagged bHLH87 proteins (such as Myc-bHLH87) to confirm antibody specificity .

• Subcellular localization confirmation: Use immunofluorescence to verify nuclear localization of bHLH87, which should align with confocal microscopy data from GFP-tagged bHLH87 studies .

• Cross-species reactivity testing: If working across plant species, test antibody recognition of bHLH87 from different species to establish cross-reactivity potential.

What are the optimal protocols for using bHLH87 antibodies in Co-immunoprecipitation (Co-IP) assays?

For effective Co-IP experiments with bHLH87 antibodies:

• Sample preparation:

  • Extract nuclear proteins from plant tissues using a nuclear isolation buffer

  • Include protease inhibitors to prevent degradation

  • For interaction studies (e.g., with pathogen effectors like SJP39), co-express both proteins in a system like N. benthamiana

• Immunoprecipitation procedure:

  • Pre-clear lysates with protein A/G beads

  • Incubate cleared lysates with bHLH87 antibody (4-10 μg) for 3-4 hours at 4°C

  • Add protein A/G beads and incubate for additional 1-2 hours

  • Wash thoroughly (at least 4 times) with wash buffer similar to that used in the SJP39-bHLH87 interaction studies (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM NaCl, 1 mM DTT)

• Detection methods:

  • For tagged proteins, use anti-tag antibodies (e.g., anti-Myc, anti-GFP)

  • For untagged proteins, use bHLH87-specific antibodies

  • Follow a protocol similar to that used in the SJP39 study, where bHLH87-Myc was detected using anti-Myc antibody after immunoprecipitation with anti-GFP beads

• Controls:

  • Include IgG controls to assess nonspecific binding

  • Use samples expressing only one protein of the interaction pair

  • Consider truncated protein variants that do not interact (e.g., SJP39 32-77 that doesn't interact with bHLH87)

How can bHLH87 antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments?

For ChIP experiments investigating bHLH87 binding to target promoters:

• Crosslinking and chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10-15 minutes

  • Isolate nuclei and sonicate chromatin to fragments of ~200-500 bp

  • Consider GA pathway genes as potential targets, based on findings that bHLH87 regulates GA-related genes like ZjKAO and ZjGRP11

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with bHLH87 antibody overnight at 4°C

  • Include appropriate controls (IgG, input samples)

  • Wash thoroughly and reverse crosslinks

• Target gene analysis:

  • Analyze enrichment of GA-pathway genes (biosynthesis and responsive genes)

  • Focus on genes identified in transcriptomic analyses, such as those in the diterpenoid metabolic pathway

  • Consider genes with altered expression in plants expressing SJP39 or overexpressing ZjbHLH87

• Validation approaches:

  • Verify binding using luciferase reporter assays with target promoters

  • Compare results with dual-luciferase assays performed with the ZjKAO or ZjGRP11 promoters

How do different treatments affect bHLH87 expression and what implications does this have for antibody detection?

Based on research findings, bHLH87 expression changes significantly under various conditions:

These expression changes have important implications for antibody-based detection:

• Sample timing is crucial after treatments

• Expression level differences may require adjustment of antibody dilutions

• Background controls should match treatment conditions

• Consider protein extraction efficiency from different tissue states

How can I distinguish between native bHLH87 and its stabilized form when interacting with pathogen effectors?

Distinguishing between native and stabilized bHLH87 requires careful experimental design:

• Quantitative Western blot analysis:

  • Compare bHLH87 levels in the presence and absence of interacting proteins like SJP39

  • Use gradient gels to detect potential mobility shifts

  • Include time-course experiments to track accumulation dynamics

• Protein stability assays:

  • Perform cycloheximide chase assays to compare bHLH87 degradation rates

  • Measure half-life in the presence and absence of stabilizing factors

  • Consider proteasome inhibitors to determine if stabilization involves protection from 26S proteasomal degradation

• Phosphorylation state analysis:

  • Use phospho-specific antibodies if phosphorylation sites are known

  • Perform phosphatase treatments to detect mobility shifts related to phosphorylation

  • Consider mass spectrometry analysis to identify post-translational modifications

• Cellular fractionation:

  • Compare nuclear vs. cytoplasmic distribution using appropriate fractionation controls

  • Quantify nuclear accumulation similar to the confocal microscopy approach used in SJP39 studies

How can bHLH87 antibodies be used to investigate the molecular mechanisms of plant disease resistance?

To investigate bHLH87's role in disease resistance:

• Pathogen infection time-course studies:

  • Track bHLH87 protein levels before and after pathogen challenge

  • Compare resistant and susceptible plant varieties as demonstrated in pumpkin lines with differential powdery mildew resistance

  • Correlate bHLH87 protein levels with disease severity indices

• Protein-protein interaction networks:

  • Use bHLH87 antibodies in Co-IP followed by mass spectrometry to identify novel interactors

  • Compare interaction partners between healthy and infected tissues

  • Focus on potential interactions with pathogen effectors like SJP39

• Hormone signaling pathway crosstalk:

  • Investigate bHLH87 involvement in hormone-mediated defense by analyzing protein levels after hormone treatments

  • Focus on GA, ABA, and JA pathways, which have demonstrated connections to bHLH87

  • Use bHLH87 antibodies to track protein accumulation after hormone treatments

• Cell death and ROS accumulation mechanisms:

  • Analyze bHLH87 in relation to cell death markers and H₂O₂ accumulation

  • Correlate with findings that CmbHLH87 expression accelerates cell necrosis and enhances H₂O₂ accumulation

What approaches can resolve contradictory findings between bHLH87 transcript and protein levels?

To address potential discrepancies between transcript and protein data:

• Simultaneous analysis of transcript and protein:

  • Perform RT-qPCR and Western blot from the same samples

  • Track both measurements across experimental treatments and time points

  • Look for time delays between transcript changes and protein accumulation

• Protein stability assessment:

  • Consider post-translational regulation mechanisms, particularly in light of findings that SJP39 stabilizes bHLH87 protein without necessarily affecting transcript levels

  • Use proteasome inhibitors (MG132) to determine if protein stability rather than transcription explains discrepancies

• Translation efficiency analysis:

  • Perform polysome profiling to assess translation of bHLH87 mRNA

  • Consider the use of translation inhibitors in time-course experiments

• Tissue-specific expression patterns:

  • Use immunohistochemistry with bHLH87 antibodies to determine if tissue-specific protein accumulation differs from whole-tissue transcript measurements

  • Consider microdissection approaches for more precise analysis

What are common challenges when using bHLH87 antibodies and how can they be addressed?

Researchers commonly encounter these issues:

• Low signal intensity:

  • Consider bHLH87 expression levels under your experimental conditions

  • Increase antibody concentration or sample loading

  • Use more sensitive detection methods (e.g., chemiluminescence substrates with longer exposure times)

  • Consider enrichment by nuclear fractionation, as bHLH87 is primarily nuclear

• Multiple bands or nonspecific binding:

  • Increase blocking stringency (5% BSA instead of standard blocking)

  • Optimize antibody dilution and incubation conditions

  • Include competing peptides to confirm specificity

  • Use bHLH87 overexpression or knockout samples as controls

• Cross-reactivity with other bHLH family members:

  • Perform pre-adsorption with recombinant proteins from related bHLH family members

  • Use epitope-specific antibodies targeting unique regions outside the conserved bHLH domain

  • Validate with genetic controls (overexpression, knockout)

• Inconsistent IP efficiency:

  • Optimize lysis conditions to ensure complete nuclear protein extraction

  • Consider crosslinking approaches for transient interactions

  • Test different antibody amounts and incubation times

  • Use tagged versions of bHLH87 as positive controls for troubleshooting

How can I optimize bHLH87 antibody performance across different plant species?

To optimize cross-species antibody performance:

• Epitope conservation analysis:

  • Align bHLH87 sequences from target species to identify conserved regions

  • Select antibodies targeting highly conserved epitopes for cross-species applications

  • Consider custom antibodies against conserved peptides if commercial options show poor cross-reactivity

• Extraction buffer optimization:

  • Adjust extraction conditions for different plant tissues and species

  • For recalcitrant species, consider more stringent extraction buffers with higher detergent concentrations

  • Optimize nuclear extraction protocols based on species-specific characteristics

• Validation in each species:

  • Use transgenic controls when possible (overexpression lines like those described for CmbHLH87)

  • Consider using epitope-tagged versions of bHLH87 from each species for initial validation

  • Establish species-specific positive and negative controls

• Protocol adjustments:

  • Modify antibody concentration, incubation time, and temperature for each species

  • Establish species-specific blocking conditions to minimize background

  • Document optimal conditions for each species in a standardized protocol