BHLH144 Antibody

What is bHLH144 and why is it important in plant molecular biology?

bHLH144 (LOC_Os04g35010) is a seed-specifically transcribed basic helix-loop-helix transcription factor that forms a heterotrimer complex with NF-YB1 and NF-YC12 in rice. This complex plays a crucial role in regulating grain quality by activating the Waxy (Wx) gene, which controls amylose content in rice endosperm. The significance of bHLH144 lies in its involvement in a sequential protein interaction where NF-YB1 binds with NF-YC12 first, and then this heterodimer interacts with bHLH144 to form a functional transcriptional complex . This interaction pattern has been verified through multiple experimental approaches including yeast-three-hybrid (Y3H), in vitro pull-down assays, and bimolecular fluorescence complementation (BiFC). Understanding bHLH144's function provides valuable insights into transcriptional regulation mechanisms in seed development and presents opportunities for rice genetic improvement .

How does bHLH144 contribute to the stability of transcription factor complexes?

bHLH144 significantly enhances the stability of NF-YB1 protein within the NF-YB1-YC12-bHLH144 complex. Cell-free degradation assays have demonstrated that NF-YB1 has a half-life of approximately 15 minutes when isolated, but this extends to 40 minutes when in complex with NF-YC12 and bHLH144 . Research indicates that NF-YB1 undergoes degradation via the ubiquitin/26S proteasome pathway, and the presence of both NF-YC12 and bHLH144 protects NF-YB1 from this degradation mechanism. While bHLH144 doesn't appear to affect NF-YB1's DNA binding capacity to the Wx promoter, its role in enhancing complex stability is crucial for maintaining transcriptional activity . This stabilizing function represents an important regulatory mechanism for controlling gene expression during seed development.

What expression patterns should researchers expect for bHLH144?

bHLH144 exhibits a seed-specific expression pattern similar to its interacting partners NF-YB1 and NF-YC12 . This tissue-specific expression is consistent with its proposed role in regulating grain development genes. When conducting immunohistochemistry or in situ hybridization experiments, researchers should anticipate nuclear localization of bHLH144, as confirmed by BiFC experiments showing that the NF-YB1-YC12-bHLH144 complex co-localizes in the nucleus . For experimental planning, it's important to note that bHLH144 expression is developmentally regulated during seed maturation, suggesting that timing of tissue collection is critical for successful detection with antibodies. Quantitative RT-PCR data indicates that expression levels can be normalized to ubiquitin using the 2^-ΔΔCT method for accurate quantification .

What criteria should guide selection of a bHLH144 antibody?

When selecting a bHLH144 antibody, researchers should first consider the experimental application, as different techniques (Western blotting, immunoprecipitation, immunohistochemistry) may require antibodies with different properties. The epitope selection is critical—antibodies raised against unique regions of bHLH144 will minimize cross-reactivity with other bHLH family members, which share conserved domains. Based on approaches used for similar transcription factor antibodies, researchers should evaluate clonality options: monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies can provide stronger signals by recognizing multiple epitopes . For studying the NF-YB1-YC12-bHLH144 complex, consider whether the antibody epitope might interfere with protein-protein interaction sites. Validation data should demonstrate antibody specificity in rice tissues, ideally with appropriate knockout controls (bhlh144 mutants) produced using CRISPR/Cas9 or similar gene editing techniques .

How should researchers validate a bHLH144 antibody?

Comprehensive validation of a bHLH144 antibody requires multiple approaches. First, perform Western blot analysis using recombinant HIS-bHLH144-FLAG protein as a positive control to confirm that the antibody recognizes the target at the expected molecular weight . Next, compare wild-type rice samples with bhlh144 knockout or knockdown lines to verify specificity—the signal should be absent or significantly reduced in mutant tissues. For co-immunoprecipitation experiments, validate that the antibody can precipitate both bHLH144 and its known interacting partners (NF-YB1 and NF-YC12) from rice seed extracts. In immunohistochemistry applications, staining should match the known nuclear localization pattern and be enriched in seed tissues consistent with transcription data . Cross-reactivity testing with related bHLH family proteins will ensure the antibody is truly specific to bHLH144. Documentation of these validation steps is essential for publication and reproducibility.

What controls are essential when working with bHLH144 antibodies?

Several controls are critical when working with bHLH144 antibodies to ensure experimental validity. Positive controls should include recombinant HIS-bHLH144-FLAG protein for Western blots and immunoprecipitation validation . Tissue samples from developing rice seeds (where bHLH144 is naturally expressed) serve as physiological positive controls. Negative controls should include tissue samples from bhlh144 knockout or knockdown plants generated using CRISPR/Cas9 methods . For immunoprecipitation specificity, pre-immune serum controls or isotype-matched control antibodies should be employed. Loading controls for Western blots typically include housekeeping proteins like ubiquitin, which has been established as a reliable normalization standard in rice seed tissues . When performing immunofluorescence studies, DAPI counterstaining should be used to verify nuclear localization, similar to the approach used for ARNT/HIF-1 beta antibody validation .

What is the recommended protocol for Western blot analysis with bHLH144 antibodies?

For Western blot analysis of bHLH144, prepare protein extracts from rice developing seeds using a buffer containing protease inhibitors to prevent degradation. Based on protocols for similar nuclear proteins, proteins should be separated on 10% SDS-PAGE gels as bHLH144 is expected to migrate at approximately 40-45 kDa. After transferring to PVDF membrane (preferred over nitrocellulose for nuclear proteins), block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature . Incubate with primary bHLH144 antibody at a concentration of 2-5 μg/mL (preliminary titration experiments may be needed) overnight at 4°C . After washing, apply HRP-conjugated secondary antibody matching the host species of the primary antibody. For enhanced sensitivity when working with low-abundance transcription factors like bHLH144, consider using chemiluminescent detection systems. Include appropriate reducing conditions (typically using DTT or β-mercaptoethanol) and reference the Immunoblot Buffer Group 1 system, which has been successfully employed for other transcription factor antibodies .

How can immunofluorescence be optimized for bHLH144 detection in plant tissues?

For optimal immunofluorescence detection of bHLH144 in rice tissues, fixation is a critical first step. Based on protocols for nuclear transcription factors, fix tissue samples in 4% paraformaldehyde for 30 minutes, followed by permeabilization with 0.2% Triton X-100 to facilitate antibody access to nuclear antigens. Antigen retrieval may be necessary for formaldehyde-fixed plant tissues—consider using citrate buffer (pH 6.0) heated to 95°C for 20 minutes. When staining, use the bHLH144 primary antibody at 5-10 μg/mL and incubate for at least 3 hours at room temperature or overnight at 4°C . For visualization, apply fluorescently conjugated secondary antibodies matching the host species of your primary antibody, with NorthernLights™ 557-conjugated antibodies providing excellent signal-to-noise ratio . Counterstain nuclei with DAPI (4′,6-diamidino-2-phenylindole) to confirm co-localization, as bHLH144 should demonstrate nuclear localization based on BiFC experiments . Image samples using confocal microscopy with appropriate filter sets for your fluorophores, focusing on rice endosperm and embryo tissues where bHLH144 expression is highest.

What are the key considerations for co-immunoprecipitation of the NF-YB1-YC12-bHLH144 complex?

When performing co-immunoprecipitation (Co-IP) to study the NF-YB1-YC12-bHLH144 complex, several factors require careful attention. First, extract preparation is critical—use a gentle lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40) supplemented with protease inhibitors to preserve protein-protein interactions . Since the formation of the trimeric complex occurs in a sequential manner (NF-YB1 binds NF-YC12 first, then bHLH144), ensure complete extraction of nuclear proteins. Pre-clear lysates with protein A/G beads to reduce non-specific binding. When using bHLH144 antibodies for immunoprecipitation, be aware that the epitope location could potentially interfere with protein-complex formation; therefore, consider multiple antibodies targeting different regions of bHLH144. Alternatively, use FLAG-tagged or His-tagged recombinant proteins and corresponding affinity beads for pull-down assays . Include RNase treatment controls to ensure interactions are not RNA-dependent. For detection of all three complex components, perform Western blotting using specific antibodies against NF-YB1, NF-YC12, and bHLH144. The sequential nature of complex formation means that pull-down with bHLH144 antibody should co-precipitate both NF-YB1 and NF-YC12 .

How should researchers troubleshoot non-specific binding with bHLH144 antibodies?

Non-specific binding is a common challenge when working with transcription factor antibodies like those for bHLH144. If experiencing high background or multiple bands in Western blots, first optimize blocking conditions by testing different blocking agents (5% BSA may be more effective than milk for some antibodies) and increasing blocking time to 2 hours. Titrate antibody concentrations systematically to determine the optimal signal-to-noise ratio—starting with a concentration of 1-2 μg/mL and adjusting as needed . For high background in immunohistochemistry, increase the number and duration of wash steps, and consider adding 0.1% Tween-20 to wash buffers. If cross-reactivity with other bHLH family members is suspected, pre-absorb the antibody with recombinant proteins of closely related bHLH transcription factors. For persistent multiple bands, validate which band represents bHLH144 by comparing wild-type samples with bhlh144 knockout tissues. Adding denaturing agents like SDS or urea during sample preparation can help reduce non-specific protein aggregation. Finally, consider testing the antibody under non-reducing conditions, as some epitopes are sensitive to reducing agents .

What approaches can be used to study bHLH144 protein stability and degradation?

To investigate bHLH144 protein stability and its role in complex formation, researchers can employ several approaches. Cell-free degradation assays, as used for NF-YB1, are particularly valuable—incubate purified HIS-bHLH144-FLAG with total protein extracts from rice seedlings and monitor degradation over time by Western blot . To determine if bHLH144 is regulated by the 26S proteasome pathway, include the proteasome inhibitor MG132 in parallel samples—if degradation is reduced, this suggests proteasomal regulation . For in vivo studies, treat rice seedlings or callus with MG132 or other proteasome inhibitors and monitor bHLH144 accumulation. Cycloheximide chase assays can distinguish between effects on protein stability versus synthesis—treat samples with cycloheximide to block new protein synthesis, then track bHLH144 levels over time with and without proteasome inhibitors. To study how NF-YB1 and NF-YC12 affect bHLH144 stability, compare degradation kinetics of bHLH144 alone versus in the presence of one or both partners . For advanced studies, ubiquitination assays using immunoprecipitation followed by ubiquitin Western blot can identify post-translational modifications that target bHLH144 for degradation.

How can researchers use bHLH144 antibodies for chromatin immunoprecipitation (ChIP) studies?

For chromatin immunoprecipitation (ChIP) studies of bHLH144 binding to genomic targets, several methodological considerations are essential. Begin with appropriate crosslinking—for plant tissues, 1% formaldehyde for 10-15 minutes under vacuum is typically effective for fixing transcription factors to DNA. Sonicate chromatin to generate fragments of 200-500 bp, checking fragment size by agarose gel electrophoresis. Since the NF-YB1-YC12-bHLH144 complex has been shown to bind the Wx promoter, this region serves as a positive control for ChIP optimization . Use 2-5 μg of bHLH144 antibody per immunoprecipitation reaction, ensuring the antibody epitope is accessible when bHLH144 is bound to DNA. A critical validation step is to perform parallel ChIP with pre-immune serum or IgG as a negative control, and potentially with NF-YB1 antibody as a positive control since these factors bind the same promoter regions . When analyzing ChIP-qPCR data, calculate enrichment relative to input DNA and normalize to a non-bound control region. For genome-wide binding studies (ChIP-seq), ensure sufficient sequencing depth (minimum 10-20 million uniquely mapped reads) and include appropriate bioinformatic analysis to identify binding motifs. Consider dual cross-linking with disuccinimidyl glutarate (DSG) followed by formaldehyde to better capture indirect DNA interactions through protein complexes.

How has bHLH144 been studied in the context of rice grain quality regulation?

bHLH144 research has revealed its crucial role in the transcriptional regulation of rice grain quality. Studies have successfully employed multiple complementary techniques to characterize its function. Yeast-three-hybrid (Y3H) systems have been instrumental in identifying the sequential interactions between NF-YB1, NF-YC12, and bHLH144, showing that NF-YB1 and NF-YC12 must first form a heterodimer before interacting with bHLH144 . Pull-down assays using glutathione agarose beads and FLAG agarose beads confirmed these interactions in vitro, while bimolecular fluorescence complementation (BiFC) verified them in planta . Genetic studies using CRISPR/Cas9-generated bhlh144 mutants demonstrated phenotypes similar to nf-yb1 and nf-yc12 mutants in seed development, supporting their joint function . Electrophoretic mobility shift assays (EMSA) revealed that while bHLH144 itself doesn't bind to the Wx promoter, it enhances the function of the complex through protein stabilization . Gene expression analysis using RNA extraction, qRT-PCR, and mRNA in situ hybridization has confirmed the seed-specific co-expression of these three transcription factors. Together, these approaches have established a model where the NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain quality in rice.

What techniques can be combined with bHLH144 antibodies for comprehensive protein function studies?

A multi-technique approach incorporating bHLH144 antibodies can provide comprehensive insights into its functional mechanisms. Start with immunoprecipitation coupled with mass spectrometry (IP-MS) to identify novel interacting partners beyond the known NF-YB1 and NF-YC12 associations . Complement this with proximity labeling methods like BioID or APEX, where bHLH144 is fused to a biotin ligase to label proximal proteins in vivo. For temporal dynamics of complex formation, use real-time live cell imaging with fluorescently-tagged proteins. Investigate chromatin interaction landscapes using CUT&RUN or CUT&Tag, which offer higher signal-to-noise ratios than traditional ChIP for transcription factors . Combine these genomic approaches with RNA-seq in wild-type and bhlh144 mutant backgrounds to correlate binding events with transcriptional outcomes. For protein-DNA interaction kinetics, employ microscale thermophoresis (MST) or bio-layer interferometry with purified components. To understand developmental context, use tissue-specific or inducible expression systems coupled with phenotypic analysis. For post-translational modification mapping, combine immunoprecipitation with phospho-proteomic or ubiquitin-proteomic analysis. This integrated approach will provide mechanistic understanding of how bHLH144 contributes to transcriptional regulation within its complex.

What are the current challenges and future directions in bHLH144 research?

Current challenges in bHLH144 research include distinguishing its specific functions from other bHLH family members, understanding the dynamics of complex assembly in different cellular contexts, and determining how environmental factors influence complex formation and stability. The sequential nature of the NF-YB1-YC12-bHLH144 complex formation presents technical challenges for structural studies, as intermediates may be unstable . Future research directions should focus on resolving the three-dimensional structure of the complete NF-YB1-YC12-bHLH144 complex bound to DNA, possibly using cryo-electron microscopy. Investigating whether NF-YA proteins also participate in this complex remains important, as current Y3H systems have been limited by NF-YA autoactivation issues . Exploring the broader regulatory network controlled by this complex beyond the Wx gene would provide insights into its comprehensive role in rice development. Comparative studies across different cereal crops could reveal evolutionary conservation of this regulatory mechanism. Development of small molecule modulators of complex formation would enable temporal control in experimental systems. Additionally, leveraging CRISPR/Cas9 precision editing to modify specific interaction domains could create separation-of-function mutants that maintain some aspects of bHLH144 activity while disrupting others, helping to dissect its multifaceted roles in plant development .