BEE1 Antibody
Description
Introduction
Bee1 protein, found in budding yeast, demonstrates a notable sequence similarity to the Wiskott-Aldrich syndrome protein (WASP), a human protein potentially linking signaling pathways to the actin cytoskeleton . Mutations in WASP are known to cause Wiskott-Aldrich syndrome, which is characterized by immunodeficiencies and defects in blood cell morphogenesis . Research indicates Bee1 protein is a crucial component of a cytoskeletal complex that regulates the assembly and organization of actin filaments at the cell cortex .
BEE1 Function and Structure
Bee1p is a component of cortical actin patches and is essential for the organization of actin filaments . Disruption of the BEE1 gene results in a striking change in the organization of actin filaments, leading to defects in budding and cytokinesis . In Δbee1 cells, actin filaments form aberrant bundles in the buds that do not contain most of the cortical cytoskeletal components, rather than assembling into cortically associated patches . Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function .
Impact on Budding and Cytokinesis
A knockout construct, in which 84% of the BEE1 coding region was replaced by the LEU2 gene, was transformed into a Leu2 − diploid yeast strain . Following sporulation and tetrad dissection, analysis revealed that all tetrads had two colonies that grew at a wild-type rate and two colonies that grew much more slowly when grown at room temperature (Figure 1A) . Approximately 37% of Δbee1 cells had more than one nucleus . A significant fraction of the cells (∼24%, compared to only 3% in wild-type population) were large budded with divided nuclei . There were also clumps of cells that could not be separated with a microdissection needle, suggesting that the Δbee1 mutation may affect budding and cytokinesis .
To assess the Δbee1 deficiency, the rates of bud emergence, bud growth, and cytokinesis were compared between Δbee1 and wild-type cells .
LIBRA-seq Technology
LIBRA-seq (Linking B-cell Receptor to Antigen Specificity through sequencing) is a technique that allows researchers to map the unique sequence of amino acids that make up the reactive portion of an antibody and match it to the specificities of the antigen it targets simultaneously and in a high throughput way . Using LIBRA-seq, researchers discovered an “ultrapotent” monoclonal antibody that recognized multiple variants of SARS-CoV-2 in 2021 . In 2023, they reported the discovery of cross-reactive antibodies that simultaneously target HIV and hepatitis C virus (HCV) .
APE1 Inhibitors
APE1 (AP endonuclease 1) is a critical protein in base excision repair (BER) that processes potentially cytotoxic abasic sites (AP sites) . APE1 is a promising new target in cancer . Several specific APE1 inhibitors were isolated, with the IC50 for APE1 inhibition ranging between 30 nM and 50 μM . APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines .
To estimate IC50 for APE1 inhibition, the ability of the compounds to inhibit APE1 at a range of concentrations (10 nM–100 μM) were evaluated in black 384-well plates . The data was fitted to a sigmoidal dose-response model using Graphpad Prism 3.0, and IC 50 values were determined using the formula: % Activity=100/(1+10 (log[I]−log IC 50)) .
Product Specs
Target Background
Q&A
What is BEE1 and why is it significant for research?
BEE1 (BR ENHANCED EXPRESSION 1) functions as a direct target of BES1 (BRI1-EMS-SUPPRESSOR 1), which is a key regulator in the brassinosteroid (BR) pathway that promotes plant growth. BEE1 acts as a positive regulator of photoperiodic flowering in plants like Arabidopsis thaliana. It binds directly to the FT chromatin to activate the transcription of FT and promote flowering initiation. Furthermore, BEE1 promotes flowering in a blue light photoreceptor CRYPTOCHROME 2 (CRY2) partially dependent manner, as it physically interacts with CRY2 under blue light .
In yeast, Bee1 is a protein that exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that links signaling pathways to the actin cytoskeleton. Disruption of BEE1 in yeast causes striking changes in actin filament organization, resulting in defects in budding and cytokinesis. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain-containing protein implicated in actin assembly and function .
What are the primary applications of BEE1 antibodies in research?
BEE1 antibodies are valuable tools for studying:
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Plant hormone signaling pathways, particularly brassinosteroid signaling
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Photoperiodic flowering control mechanisms
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Blue light signaling in plants
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Actin cytoskeleton organization in yeast
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Protein-protein interactions involving BEE1/Bee1
These antibodies can be used in various experimental techniques including Western blotting, immunoprecipitation, immunohistochemistry, and chromatin immunoprecipitation (ChIP) assays to detect and analyze BEE1 expression, localization, and interactions with other proteins.
How should I choose between polyclonal and monoclonal BEE1 antibodies?
When selecting between polyclonal and monoclonal BEE1 antibodies, consider these research-specific factors:
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Polyclonal antibodies: Recognize multiple epitopes on BEE1, offering higher sensitivity but potentially lower specificity. These are ideal for initial detection studies or when protein conformation may vary.
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Monoclonal antibodies: Target a single epitope, providing higher specificity but potentially lower sensitivity. These are preferred for distinguishing between closely related proteins or when reproducibility across experiments is critical.
Recent demonstrations by organizations like YCharOS and Abcam using KO cell lines have shown that recombinant antibodies are more effective than polyclonal antibodies and far more reproducible . This is particularly important for long-term research projects requiring consistent antibody performance.
What are the optimal methods for validating BEE1 antibody specificity?
Validating BEE1 antibody specificity is critical for ensuring experimental reliability. The International Working Group for Antibody Validation established "five pillars" of antibody characterization:
| Validation Method | Description | Advantages | Limitations |
|---|---|---|---|
| Genetic strategies | Using knockout/knockdown of BEE1 as controls | Gold standard for specificity | Requires genetic manipulation capability |
| Orthogonal strategies | Comparing antibody-dependent and antibody-independent experiments | Confirms results through multiple methods | May require different experimental expertise |
| Independent antibody strategies | Using different antibodies targeting the same protein | Increases confidence in target identification | Requires multiple validated antibodies |
| Recombinant strategies | Increasing target protein expression | Confirms signal increases with expression | May not reflect native conditions |
| Immunocapture MS strategies | Using mass spectrometry to identify captured proteins | Directly identifies bound proteins | Requires specialized equipment |
For BEE1 antibodies, comprehensive validation should document: (i) that the antibody binds to the target protein; (ii) that it binds to BEE1 in complex protein mixtures; (iii) that it doesn't bind to non-target proteins; and (iv) that it performs as expected in specific experimental conditions .
How can I optimize Western blot protocols for BEE1 detection?
For optimal Western blot detection of BEE1:
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Sample preparation: For plant tissues, use a buffer containing protease inhibitors to prevent degradation. For yeast samples, use appropriate lysis methods to ensure complete protein extraction.
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Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution of BEE1 (expected molecular weight of approximately 60 kDa in plants).
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Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight at 4°C.
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Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.
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Primary antibody incubation: Dilute BEE1 antibody at 1:1000 in blocking buffer and incubate overnight at 4°C .
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Secondary antibody selection: Choose a species-appropriate HRP-conjugated secondary antibody. For example, if using a rabbit-derived primary BEE1 antibody, use an anti-rabbit secondary antibody .
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Controls: Always include positive and negative controls, such as extracts from BEE1 overexpression and knockout lines, to validate specificity.
What considerations are important for immunoprecipitation using BEE1 antibodies?
When performing immunoprecipitation with BEE1 antibodies:
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Antibody amount: Use approximately 1:100 dilution for immunoprecipitation applications .
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Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.
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Cross-linking: Consider cross-linking the antibody to beads to prevent antibody co-elution with target proteins.
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Washing conditions: Use stringent washing conditions (high salt, detergents) to reduce background while maintaining specific interactions.
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Elution method: Choose between native elution (maintaining protein-protein interactions) or denaturing elution (higher yield but disrupts complexes).
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Validation: Confirm successful immunoprecipitation by Western blotting a portion of the eluate.
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Interaction studies: For studying BEE1 interactions (such as with CRY2 or other pathway components), consider co-immunoprecipitation under blue light conditions to capture light-dependent interactions .
How can I apply BEE1 antibodies in chromatin immunoprecipitation (ChIP) assays?
For effective ChIP assays with BEE1 antibodies:
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Cross-linking: Cross-link proteins to DNA using 1% formaldehyde for 10 minutes at room temperature.
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Sonication: Optimize sonication conditions to produce DNA fragments of 200-500 bp.
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Antibody selection: Use ChIP-validated BEE1 antibodies; not all antibodies that work in Western blots will work in ChIP.
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Controls: Include input DNA, IgG control, and positive controls (antibodies against known DNA-binding proteins).
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Target validation: Design primers for known BEE1 binding regions, such as the FT chromatin in Arabidopsis, as BEE1 has been shown to bind directly to the FT chromatin to activate transcription .
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Data analysis: Normalize ChIP-qPCR data to input DNA and IgG control to account for background binding.
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Genome-wide studies: For ChIP-seq, ensure sufficient sequencing depth to detect binding sites across the genome.
How can BEE1 antibodies be used to study protein-protein interactions beyond standard co-IP?
Beyond standard co-immunoprecipitation, advanced techniques for studying BEE1 protein interactions include:
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Proximity Ligation Assay (PLA): This technique allows visualization of protein-protein interactions in situ with high sensitivity. For example, PLA could be used to detect BEE1-CRY2 interactions under blue light conditions in plant cells.
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FRET-based approaches: When combined with fluorescently tagged interaction partners, antibodies can help validate FRET signals.
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Pull-down assays with recombinant proteins: Use purified recombinant BEE1 in combination with candidate interacting proteins to verify direct interactions.
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Crosslinking mass spectrometry (XL-MS): This technique combines chemical crosslinking with mass spectrometry to identify protein interaction interfaces at amino acid resolution.
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Yeast two-hybrid validation: BEE1 antibodies can validate interactions identified through Y2H screens by confirming expression of fusion proteins.
When studying the BEE1-CRY2 interaction specifically, consider that it is reported to be blue light-dependent, so experimental conditions should include appropriate light treatments .
What approaches can be used to study BEE1 function in different subcellular compartments?
To investigate BEE1 localization and function in different cellular compartments:
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Subcellular fractionation: Separate cellular compartments biochemically and use BEE1 antibodies to detect the protein in different fractions.
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Immunofluorescence microscopy: Use fluorescently labeled secondary antibodies to visualize BEE1 localization in fixed cells.
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Super-resolution microscopy: For detailed localization studies, techniques like STORM or PALM provide higher resolution than conventional microscopy.
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Electron microscopy with immunogold labeling: For ultrastructural localization of BEE1.
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Live-cell imaging: While not directly using antibodies, validation of fluorescently tagged BEE1 constructs with antibodies ensures that fusion proteins behave like native BEE1.
For yeast Bee1, studies have shown localization to actin patches , while plant BEE1 may show nuclear localization consistent with its role in transcriptional regulation.
What are common issues with BEE1 antibodies and how can they be resolved?
| Issue | Possible Causes | Solutions |
|---|---|---|
| No signal in Western blot | Low BEE1 expression, antibody degradation | Increase protein loading, verify antibody storage conditions |
| Multiple bands | Cross-reactivity, protein degradation | Increase antibody dilution, add protease inhibitors, validate with KO controls |
| High background | Insufficient blocking, excessive antibody | Optimize blocking conditions, increase washing steps, dilute antibody further |
| Inconsistent results | Lot-to-lot antibody variation | Use recombinant antibodies, perform lot validation before extensive use |
| Poor immunoprecipitation | Low affinity, inappropriate buffer | Optimize antibody amount, adjust buffer conditions, consider protein A vs. G beads |
Research has estimated that ~50% of commercial antibodies fail to meet basic standards for characterization, potentially resulting in financial losses of $0.4–1.8 billion per year in the United States alone . To avoid these issues, thorough validation is essential.
How should I assess batch-to-batch variation in BEE1 antibodies?
To manage batch-to-batch variation:
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Standard sample testing: Test each new batch against a standard positive control sample.
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Side-by-side comparison: Run experiments with old and new batches simultaneously.
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Quantitative assessment: Compare signal intensity, background levels, and specificity metrics.
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Documentation: Maintain detailed records of antibody performance across batches.
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Consider recombinant alternatives: Recombinant antibodies show far less batch-to-batch variation than traditional antibodies .
When planning long-term studies, purchase sufficient quantities of a single batch or consider switching to recombinant antibodies, which have been demonstrated to be more reproducible than polyclonal antibodies in controlled studies by organizations like YCharOS and Abcam .
How can I determine if my BEE1 antibody recognizes native or denatured protein forms?
To determine if your BEE1 antibody recognizes native or denatured forms:
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Compare different techniques:
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Western blotting (denatured protein)
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Native PAGE (non-denatured)
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Immunoprecipitation (generally native)
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ELISA (can be adapted for both forms)
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Epitope mapping: If known, the location of the epitope can predict whether the antibody recognizes native or denatured forms.
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Direct comparison experiment: Test antibody against both native protein samples and heat/SDS-denatured samples.
This distinction is critical because studies have shown that antibodies like those against Bet v 1 can exhibit dramatically different binding properties to folded versus unfolded proteins. For instance, results from indicated that "IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1, and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes."
How can emerging antibody technologies be applied to BEE1 research?
Recent technological advances offer new opportunities for BEE1 research:
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Recombinant antibody generation: Tools like RFdiffusion can be used to design human-like antibodies, including those targeting specific epitopes of BEE1. This AI-driven approach produces antibody blueprints that bind specified targets .
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Bispecific antibodies: New platforms like PHE-Ig technique promote desired chain pairing by replacing CH1/CL regions with natural A and B chains of PHE1 fragment based on knob-in-hole technology. This approach could be adapted to create BEE1-targeting bispecific antibodies for complex applications .
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Antibody fragments: Single-chain variable fragments (scFvs) offer improved tissue penetration for in vivo studies of BEE1 function.
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Site-specific labeling: Next-generation antibody conjugation methods allow precise control over label location, improving consistency in imaging and detection.
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Nanobodies: These single-domain antibody fragments derived from camelids offer advantages of small size and stability for certain applications.
What computational approaches can improve BEE1 antibody design and selection?
Computational methods are increasingly important in antibody research:
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Epitope prediction: Algorithms can predict likely epitopes on BEE1, guiding antibody design toward regions most likely to yield specific antibodies.
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Biophysics-informed modeling: Advanced models can predict binding properties and help generate antibody variants with desired specificity profiles. For example, research has demonstrated "the computational design of antibodies with customized specificity profiles, either with specific high affinity for a particular target ligand, or with cross-specificity for multiple target ligands" .
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Machine learning for antibody validation: ML algorithms can help predict antibody performance based on sequence and structure information.
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Structural modeling: Techniques like AlphaFold can predict BEE1 structure and potential antibody binding sites.
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Affinity optimization: Computational protein design tools can suggest mutations to improve antibody affinity and specificity.
How can large-scale antibody characterization initiatives inform BEE1 antibody selection?
Large-scale characterization initiatives provide valuable resources:
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YCharOS and validation standards: YCharOS assessments using knockout cell lines have demonstrated that recombinant antibodies are more effective and reproducible than polyclonal antibodies .
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The "five pillars" framework: The International Working Group for Antibody Validation established standards that should guide BEE1 antibody validation:
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RRID system implementation: Using Research Resource Identifiers (RRIDs) to track antibody use across publications can help identify reliable BEE1 antibodies based on successful use in multiple studies.
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Data sharing platforms: Repositories of antibody validation data allow researchers to make informed decisions based on comprehensive characterization data.
For BEE1 antibodies specifically, researchers should consult these resources before selecting antibodies for their studies to ensure reproducibility and reliability of results.
