mycb Antibody
Description
Introduction to MYCB Antibody
MYCB antibody is a research tool designed to detect endogenous levels of total MYCB protein in biological samples. These antibodies are primarily used for research purposes and are not intended for diagnostic procedures or therapeutic applications without explicit authorization . The target protein, MYCB (also known as B-Myc), functions as an inhibitor of cellular proliferation and plays important roles in various biological processes .
MYCB protein is known by several aliases, including:
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Avian myelocytomatosis viral (v-myc) related oncogene
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Bmyc protein (AA 1-178)
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Protein B-Myc
The gene encoding MYCB is also referenced under multiple names, including 2900002K07Rik, AW060705, B-myc, Bmyc, and Mycb, with UniProt IDs Q6P8Z1 (Mouse) and P15063 (Rat) .
MYCB Antibody Characteristics
Commercial MYCB antibodies, such as the Rat Mycb Antibody (catalog number 60-108), are typically produced as polyclonal antibodies in rabbits immunized with synthetic peptides derived from the C-terminal region of rat Mycb . These antibodies are purified through protein A column chromatography followed by peptide affinity purification . They have a predicted molecular weight of approximately 18 kDa and are supplied in PBS buffer with 0.09% sodium azide .
MYCB Protein Structure
B-Myc (MYCB) is an endogenous, N-terminal homologue of the transcription factor c-Myc but lacks the C-terminal DNA binding and protein dimerization domain present in c-Myc . Nuclear magnetic resonance (NMR) spectroscopy studies have revealed that B-Myc has no persistent tertiary structure, yet regions corresponding to Myc homology boxes 1 and 2 (MBI and MBII, respectively) exhibit molten globule-like characteristics .
The carboxy-terminal half of MYC includes MB3b and MB4 domains, along with the basic helix-loop-helix (bHLH) and leucine zipper domains involved in DNA binding and MYC:MAX heterodimerization . The MB3b domain of human MYC harbors PEST and WDR5-binding domains that are conserved in Mycb and contribute to protein stability and chromatin interaction .
Within the MB4 domain is a calpain cleavage site necessary for generating a cytoplasmic form of MYC called MYC-Nick that stimulates α-tubulin acetylation and cellular differentiation . This region is largely conserved between MYC and Mycb. MB4 also contains the HCF-1 binding site, which impacts MYC-driven tumorigenesis by regulating ribosome biogenesis and mitochondrial gene expression, and this sequence is conserved in Mycb .
General Functions
MYCB functions as a transcription factor that appears to inhibit cellular proliferation, contrasting with the proliferation-promoting activity of other MYC family members . Studies show that B-Myc can interact with the transcriptional machinery and inhibit reporter gene activation by a GAL4 chimeric protein containing the c-Myc transcriptional activation domain .
When overexpressed, B-Myc dramatically inhibits the neoplastic cotransforming activity of c-Myc and activated Ras in rat embryo cells . This inhibitory effect on both neoplastic transformation and transcriptional activation suggests that the transforming activity of c-Myc is related to its ability to regulate transcription .
Role in Tissue Regeneration
Recent research has revealed that Mycb plays a significant role in tissue regeneration, particularly in the zebrafish retina . Following retinal injury, Mycb expression is stimulated in Müller glial cells (MG), where it promotes reprogramming and proliferation of these cells to generate Müller glia-derived progenitor cells (MGPCs) .
Mycb regulates approximately 40% of the regeneration-associated transcriptome in these cells, with a bias toward regulating cellular processes such as protein synthesis and cell division . Although Mycb and another MYC family member, Mych, regulate many of the same genes, they exhibit biases suggesting non-redundant functions .
Protein-Protein Interactions
B-Myc binds to MM-1 (a transactivation inhibitor) and TBP (an activator) in specific manners without becoming highly structured . The local regions of B-Myc involved in binding differ for MM-1 and TBP, and regions not previously identified by mutagenesis have been found to be involved in MM-1 binding .
In the context of zebrafish retina regeneration, Mycb works in collaboration with Hdac1 to inhibit her4.1, an effector of Delta-Notch signaling . It also exhibits a dual regulatory role on lin28a expression, functioning as an activator through Ascl1a in MGPCs and as a repressor in combination with Hdac1 in neighboring cells .
Antibody-Based Detection Methods
MYCB antibodies are valuable tools for detecting and studying MYCB protein expression in various research contexts. The primary applications include:
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Western Blotting (WB): For detecting MYCB protein in tissue lysates and cell extracts
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Immunohistochemistry (IHC): For visualizing MYCB expression and localization in tissue sections
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Immunocytochemistry (ICC): For studying MYCB in individual cells
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Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of MYCB levels
Advanced Analytical Techniques
Beyond standard antibody-based methods, more sophisticated techniques have been employed to study MYCB:
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Quantitative proteomics using isotope-coded affinity tag (ICAT) reagent labeling and tandem mass spectrometry has been used to analyze the function of Myc proteins in mammalian cells
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Surface-enhanced laser desorption ionization/time-of-flight mass spectrometry (SELDI/TOF-MS) for analyzing complex protein mixtures
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Gene expression analysis via reverse transcriptase polymerase chain reaction (RT-PCR) and oligonucleotide microarrays to study MYCB at the transcript level
Validation of Antibody Specificity
Ensuring antibody specificity is crucial for obtaining reliable research results. The European Monoclonal Antibody Network recommends a stepwise strategy for antibody validation, including testing on known positive and negative samples, using knockout cell lines, and confirming specificity across multiple techniques .
Initiatives like YCharOS at McGill University have refined approaches based on knockout cell lines to test antibodies in Western Blots, immunoprecipitation, and immunofluorescence . Their protocols can be widely used in antibody characterization efforts.
Role in Retina Regeneration
Recent studies have provided significant insights into the role of Mycb in zebrafish retina regeneration:
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Retinal injury stimulates mycb expression in Müller glial cells (MG)
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Mycb promotes MG reprogramming and proliferation, contributing to retinal regeneration
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Mycb regulates approximately 40% of the regeneration-associated transcriptome, with a bias toward cell cycle and DNA replication processes compared to Mych
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Mycb exhibits relatively high basal expression in MG under normal conditions
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Forced expression of Mycb is sufficient to stimulate MG proliferation in the uninjured retina, and this effect is enhanced by γ-secretase inhibition and by forced expression of Ascl1a and Lin28a
Mycb and Cell Growth Regulation
Studies on MYC family proteins, including MYCB, have revealed important roles in cell growth and protein synthesis:
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Overexpression of c-myc in Eμ-myc transgenic mice results in an increase in cell size across different developmental stages of B cells
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This increase in cell size correlates with a 2-fold increase in total protein content and protein biosynthesis
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c-Myc overexpression appears to uncouple growth from proliferation, with the predominant effect being on cell growth
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These findings suggest that Myc proteins, including potentially MYCB, play crucial roles in regulating cell growth and protein synthesis
Therapeutic Implications
While MYCB antibodies themselves are primarily research tools, understanding MYCB and related MYC proteins has important therapeutic implications:
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MYC oncogenes are deregulated in approximately 70% of human cancers and are associated with aggressive disease and drug resistance
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The OMO-103 mini protein, which inhibits MYC, has successfully completed a phase I clinical trial and is being evaluated in additional clinical trials for pancreatic cancer and advanced osteosarcoma
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Therapies targeting MYC-driven malignancies, such as the combination of tigecycline and venetoclax for MYC/BCL2 double-hit lymphomas, show promise in preclinical studies
Production Methods
MYCB antibodies are typically produced using one of two main approaches:
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Polyclonal antibody production: Involves immunizing animals (often rabbits) with a synthetic peptide derived from MYCB protein sequence, followed by purification steps including protein A column chromatography and peptide affinity purification
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Monoclonal antibody production: More specialized facilities like NeuroMab at the University of California Davis generate mouse monoclonal antibodies through hybridoma technology, screening approximately 1,000 clones in parallel ELISA assays against both the immunogen and transfected cells expressing the antigen of interest
Validation and Quality Control
Proper validation of MYCB antibodies is essential for ensuring reliable research results. Key validation steps include:
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Testing antibody specificity in multiple assay formats (Western blotting, immunoprecipitation, immunofluorescence)
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Using positive and negative controls, including knockout cell lines where available
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Confirming antibody performance across different sample types and experimental conditions
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Evaluating cross-reactivity with related proteins (other MYC family members)
Research Initiatives and Resources
Several major initiatives have contributed to the development and characterization of antibodies for research, including those targeting MYCB:
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The Recombinant Antibody Network (RAN), a consortium comprising research groups from UC San Francisco, the University of Chicago, and the University of Toronto, focuses on creating high-performance recombinant antibodies
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The Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa maintains and distributes hybridoma cell lines and monoclonal antibodies at minimal cost
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The European Monoclonal Antibody Network (EuroMAbNet) shares practical experience in antibody production, selection, and validation with the research community
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YCharOS at McGill University's Montreal Neurological Institute focuses on characterizing existing antibodies through standardized protocols
Emerging Applications
The continued development and characterization of MYCB antibodies will enable several emerging research applications:
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Single-cell analysis of MYCB expression in diverse tissues and disease states
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Spatial transcriptomics and proteomics to understand MYCB localization and function
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Therapeutic targeting strategies based on MYCB inhibition or regulation
Technological Advancements
Advances in antibody technology are likely to enhance MYCB research:
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Development of recombinant antibodies with improved specificity and lot-to-lot consistency
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Integration with CRISPR-based gene editing for more precise functional studies
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Improved imaging techniques for visualizing MYCB protein dynamics in living cells
Therapeutic Potential
While MYCB antibodies themselves are research tools, understanding MYCB function has therapeutic implications:
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The inhibitory effect of B-Myc on c-Myc-induced transformation suggests potential applications in cancer therapy
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The role of Mycb in tissue regeneration, particularly in retinal repair, points to possible regenerative medicine applications
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Understanding MYCB's regulatory mechanisms may lead to new therapeutic strategies for modulating cell growth and proliferation
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
What is the MYCB protein and why is it targeted in research?
MYCB (also known as B-myc) appears to function as an inhibitor of cellular proliferation based on current research findings. The protein has gained attention in developmental biology and cancer research due to its relationship to the MYC family of transcription factors. Understanding MYCB's role in cellular processes requires specific antibodies that can detect and quantify this protein in various experimental contexts . The protein's involvement in proliferation pathways makes it a valuable target for researchers investigating cell cycle regulation and oncogenesis mechanisms.
What types of MYCB antibodies are available for research applications?
Research laboratories can access several forms of MYCB antibodies, including rabbit polyclonal antibodies conjugated with HRP (Horseradish Peroxidase) that specifically target the C-terminal epitope of mouse MYCB . Additionally, recombinant monoclonal antibodies such as the EP643Y clone have been developed for detecting rat MYCB protein . These antibodies have been validated for Western blot applications with specific dilution recommendations (polyclonal antibodies at 1:100-500 for Western blot; 1:1000 for ELISA) . The choice between polyclonal and monoclonal formats depends on the specific research requirements, with monoclonal antibodies offering greater specificity while polyclonal antibodies may provide enhanced sensitivity.
What sample types can be analyzed using commercial MYCB antibodies?
Commercial MYCB antibodies have been validated for detecting MYCB protein in specific species and sample types. For instance, rabbit polyclonal antibodies show reactivity with mouse samples , while certain recombinant monoclonal antibodies like EP643Y demonstrate effectiveness with rat samples, particularly from brain tissue and PC12 cell lines . The observed molecular weight of detected MYCB protein is approximately 20 kDa, slightly higher than the predicted size of 18.3 kDa, which suggests potential post-translational modifications that researchers should consider when analyzing their results .
How should researchers validate MYCB antibodies before experimental use?
Proper validation of MYCB antibodies is essential given the widespread concerns about antibody reliability in research. An estimated 50% of commercial antibodies fail to meet basic characterization standards, potentially contributing to billions in research waste annually . For MYCB antibodies, validation should include: (1) Western blot analysis comparing positive controls (tissues known to express MYCB) with negative controls; (2) testing with recombinant MYCB protein; (3) knockdown or knockout experiments to confirm specificity; and (4) cross-reactivity testing against related proteins. Researchers should also review supplier validation data and independent literature reports rather than relying solely on commercial claims .
How do sample preparation methods affect MYCB antibody performance?
Sample preparation significantly impacts antibody performance in detecting MYCB protein. When preparing samples for Western blot analysis, researchers should consider that MYCB protein appears at approximately 20 kDa despite its predicted size of 18.3 kDa . This discrepancy suggests post-translational modifications that may be affected by sample preparation methods. Lysis buffer composition, protease inhibitor inclusion, heating conditions, and reducing agent concentration can all influence epitope accessibility. For tissue samples, fixation methods must be optimized to preserve MYCB epitopes while maintaining tissue morphology, particularly for immunohistochemistry applications.
How can researchers address cross-reactivity concerns with MYCB antibodies?
Cross-reactivity represents a significant challenge when working with MYCB antibodies due to homology with other MYC family proteins. To address this concern, researchers should implement a multi-tiered validation approach including: (1) competitive binding assays with purified MYCB protein; (2) parallel testing with multiple MYCB antibodies targeting different epitopes; (3) mass spectrometry validation of immunoprecipitated proteins; and (4) testing in cells with MYCB knockout or knockdown. Additionally, computational analysis of antibody binding sites across the MYC family can help predict potential cross-reactivity issues before experimental implementation .
How do post-translational modifications of MYCB affect antibody epitope recognition?
Post-translational modifications (PTMs) can significantly alter antibody recognition of MYCB protein. The observed molecular weight discrepancy (20 kDa observed vs. 18.3 kDa predicted) suggests the presence of PTMs that researchers must consider . Phosphorylation, glycosylation, ubiquitination, or SUMOylation may affect epitope accessibility or antibody binding affinity. Researchers investigating MYCB function should consider using modification-specific antibodies when studying regulated MYCB activity. Additionally, sample preparation methods that preserve or remove specific modifications should be selected based on research objectives. Phosphatase or deglycosylase treatments prior to immunodetection can help determine if modifications affect antibody recognition.
What are the considerations for using MYCB antibodies in multiplex immunoassays?
When incorporating MYCB antibodies into multiplex detection systems, researchers must address several technical challenges. First, antibody cross-reactivity must be thoroughly evaluated against all targets in the multiplex panel. Second, compatible detection systems must be selected—for HRP-conjugated MYCB antibodies, researchers must ensure substrate compatibility with other detection methods . Third, antibody combinations must be tested for potential interference effects. Pilot experiments should validate that MYCB detection sensitivity remains consistent in multiplex formats compared to single-target detection. Controls for each target should be included to verify that multiplex detection doesn't compromise individual target sensitivity or specificity.
What strategies can resolve inconsistent MYCB antibody performance across experiments?
Inconsistent antibody performance often stems from variable experimental conditions or reagent quality. Researchers should implement standardized protocols incorporating: (1) consistent lot numbers for critical reagents; (2) preparation of antibody aliquots to minimize freeze-thaw cycles; (3) standardized sample preparation methods; and (4) inclusion of positive controls in each experiment. Additionally, researchers should maintain detailed records of antibody performance across experiments, including images of full blots rather than cropped results. Comprehensive record-keeping facilitates troubleshooting when inconsistencies arise and contributes to the broader goal of improving research reproducibility in antibody-based methods .
How can researchers distinguish between specific and non-specific binding when using MYCB antibodies?
Distinguishing specific from non-specific binding requires rigorous controls and validation steps. Researchers should: (1) include negative controls lacking primary antibody; (2) use competing peptides to block specific binding sites; (3) compare results across multiple antibodies targeting different MYCB epitopes; and (4) validate with genetic approaches (siRNA, CRISPR) that reduce target expression. Additionally, dilution series experiments can help identify optimal antibody concentrations that maximize specific signal while minimizing background. For Western blot applications, full blot images should be examined for bands beyond the expected 20 kDa MYCB signal that might indicate non-specific binding .
What approaches can enhance MYCB detection sensitivity in samples with low expression?
For samples with low MYCB expression, several methodological enhancements can improve detection: (1) optimizing sample enrichment through immunoprecipitation prior to analysis; (2) utilizing signal amplification systems such as tyramide signal amplification for immunohistochemistry; (3) employing more sensitive detection substrates for HRP-conjugated antibodies; and (4) concentrating protein samples before Western blot analysis. Additionally, recombinant monoclonal antibodies may offer superior sensitivity and consistency compared to polyclonal alternatives . Extended exposure times may improve signal detection but should be balanced against increasing background signal that could compromise specificity.
How should researchers evaluate commercially available MYCB antibodies for experimental reliability?
Evaluating commercial MYCB antibodies requires systematic assessment before incorporation into critical experiments. Researchers should: (1) review published validation data, including full blot images and controls; (2) check citation records for successful applications in similar experimental contexts; (3) test antibodies using positive and negative controls relevant to their research model; and (4) compare performance across multiple vendors when possible. Independent validation is crucial given estimates that approximately 50% of commercial antibodies fail to meet basic characterization standards . Researchers should also consider antibody format, with recombinant monoclonal antibodies often providing superior reproducibility compared to conventional hybridoma-derived or polyclonal alternatives.
What are the most effective positive and negative controls for MYCB antibody validation?
Effective controls are essential for rigorous MYCB antibody validation. Positive controls should include samples known to express MYCB, such as rat brain tissue or PC12 cell lysates for antibodies targeting rat MYCB . Recombinant MYCB protein can serve as a defined positive control with known concentration. Negative controls should include: (1) samples from MYCB knockout models; (2) cell lines with confirmed absence of MYCB expression; (3) immunoprecipitation with non-specific IgG; and (4) antibody pre-absorption with immunizing peptide. For Western blot applications, molecular weight markers should flank samples to verify the observed 20 kDa band corresponds to expected MYCB size .
How can researchers contribute to improving MYCB antibody characterization in the scientific community?
Individual researchers play a crucial role in addressing the broader "antibody characterization crisis" . To contribute to improved MYCB antibody characterization, researchers should: (1) publish comprehensive validation data including positive and negative controls; (2) deposit detailed protocols in repositories like protocols.io; (3) specify exact antibody catalog numbers, lot numbers, and dilutions in publications; and (4) report both successful and unsuccessful applications to antibody validation databases. Additionally, when discovering performance issues with commercial antibodies, researchers should report findings to vendors and consider publishing these observations as resource papers. This collective approach to antibody validation supports reproducibility across the scientific community .
How might emerging antibody technologies improve MYCB detection specificity and reproducibility?
Emerging technologies promise to address current limitations in MYCB antibody research. Recombinant antibody production represents a significant advance over hybridoma-derived antibodies, offering improved batch-to-batch consistency and defined sequences that enhance reproducibility . Techniques like phage display and synthetic antibody libraries allow selection of high-affinity, highly-specific MYCB binders. Additionally, nanobodies and single-domain antibodies provide advantages for detecting MYCB in complex samples due to their small size and robust stability. The integration of computational approaches for epitope prediction with experimental validation will likely yield next-generation MYCB antibodies with superior performance characteristics compared to current options.
What advances in antibody engineering could enhance MYCB studies in challenging research contexts?
Advanced antibody engineering approaches offer solutions for MYCB research in challenging contexts. Site-specific conjugation technologies allow precise attachment of detection molecules without compromising binding sites, enhancing signal-to-noise ratios . Bi-specific antibodies targeting MYCB and a second protein could enable studies of protein-protein interactions in native contexts. Additionally, antibody fragments with enhanced tissue penetration properties could improve MYCB detection in three-dimensional cultures, organoids, or intact tissues. Coupling these engineered antibodies with emerging imaging technologies such as expansion microscopy or super-resolution approaches could reveal previously undetectable aspects of MYCB localization and function.
