Phospho-MYC (S62) Recombinant Monoclonal Antibody
Description
Phospho-MYC (S62) Recombinant Monoclonal Antibody: Overview
The Phospho-MYC (S62) Recombinant Monoclonal Antibody is a highly specific reagent designed to detect the phosphorylation of serine 62 (S62) on the MYC transcription factor. This post-translational modification is critical for regulating MYC’s transcriptional activity, proteasomal degradation, and its role in cellular proliferation and oncogenesis .
Production and Mechanism of Action
This antibody is generated through recombinant technology:
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Immunogen Preparation: Synthetic peptides corresponding to MYC phosphorylated at S62 are used to immunize rabbits or mice .
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Gene Cloning: Antibody genes are isolated, inserted into expression vectors, and transfected into host cells (e.g., HEK293F or suspension cultures) .
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Purification: Affinity chromatography isolates the antibody from cell supernatants, ensuring specificity .
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Validation: Assays like ELISA, Western blot (WB), and immunofluorescence (IF) confirm binding to phosphorylated MYC .
Applications and Recommended Dilutions
The antibody is versatile for detecting phosphorylated MYC in diverse experimental settings:
Biological Significance of MYC S62 Phosphorylation
Phosphorylation at S62 is a regulatory checkpoint:
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Degradation Control: S62 phosphorylation primes Thr58 phosphorylation by GSK-3, triggering MYC degradation via the proteasome .
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Transcriptional Activation: Phosphorylated MYC binds DNA motifs (e.g., 5'-CAC[GA]TG-3') to activate pro-growth genes (e.g., VEGFA) and promote angiogenesis .
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Cancer Implications: Dysregulated S62 phosphorylation is linked to oncogenesis, as MYC overexpression drives cell cycle progression and apoptosis evasion .
Research Findings and Utility
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Angiogenesis: MYC S62 phosphorylation promotes VEGFA expression, enabling tumor vascularization .
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Stem Cell Regulation: Phosphorylated MYC maintains embryonic stem cell self-renewal and reprogramming capacity .
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Splicing Regulation: MYC modulates splicing of PKM through HNRNPA and PTBP1, influencing metabolic isoform production .
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Cancer Therapeutics: Antibodies targeting MYC S62 phosphorylation are critical for studying MYC-driven cancers (e.g., Burkitt lymphoma) .
Conjugation Options for Enhanced Detection
Product Specs
The development of the phospho-MYC (S62) recombinant monoclonal antibody involves a multi-step process. Initially, genes encoding the MYC antibody are retrieved from rabbits previously exposed to a synthetic peptide derived from the human MYC protein phosphorylated at S62. These antibody genes are then integrated into specialized expression vectors. Subsequently, these vectors are introduced into host suspension cells, which are cultured to induce the expression and secretion of antibodies. Following this cultivation phase, the phospho-MYC (S62) recombinant monoclonal antibody is purified using affinity chromatography techniques, separating the antibody from the cell culture supernatant. Finally, the antibody's functionality is rigorously evaluated through various assays, including ELISA, Western blotting, and immunofluorescence tests, confirming its ability to interact with the human MYC protein phosphorylated at S62.
Phosphorylation of MYC at S62 plays a critical role in regulating MYC's transcriptional activity and function. Disruptions in this phosphorylation event can have significant implications for cancer development and progression, making MYC a crucial target for cancer research and therapy.
Target Background
- This study demonstrates that hsamiR24 suppresses metastasis in nasopharyngeal carcinoma by regulating the cMyc/EMT axis, suggesting that hsamiR24 could serve as a prognostic factor and a novel target for the prevention of nasopharyngeal carcinoma metastasis. PMID: 30226609
- lncRNA THOR is up-regulated in retinoblastoma, and its over-expression significantly enhances the malignant phenotype transformation of retinoblastoma cells by up-regulating c-myc and TGF2BP1 expression. PMID: 30119193
- Our findings demonstrate that neither MYC IHC nor MYC FISH alone is sufficient for identifying the clinically relevant entities of HGBLwR or DEL. PMID: 28868942
- Since RPL23 is encoded by a target gene of c-Myc, the RPL23/Miz-1/c-Myc regulatory circuit provides a feedback loop that links efficient RPL23 expression with c-Myc's function to suppress Miz-1-induced Cdk inhibitors, thereby leading to apoptotic resistance in higher-risk myelodysplastic syndrome patients. PMID: 28539603
- GATAD2B interacts with C-MYC to enhance KRAS driven tumor growth. PMID: 30013058
- Low expression of c-Myc protein predicts poor outcomes in patients with HCC undergoing hepatectomy. PMID: 29690860
- Collectively, these findings suggest that c-Myc could transcriptionally regulate TCRP1 in cell lines and clinical samples and identified the c-Myc-TCRP1 axis as a negative biomarker of prognosis in tongue and lung cancers. PMID: 28623290
- Kazakh and Han patients with esophageal squamous cell carcinoma with Glut1 c-myc co-expression exhibited poorer prognosis. PMID: 29629851
- MYC activation in papillary clear cell renal cell carcinoma leads to a worse prognosis. PMID: 28593993
- We did not find any relationship between Bcl-2, c-Myc and EBER-ISH positivity and the low/high IPS groups in classical Hodgkin lymphoma. PMID: 29708579
- Fluorescence in situ hybridization studies (histologic sections) confirmed translocations of MYC (8q24), BCL2 (18q21) and BCL6 (3q27) in all patients. PMID: 30043475
- Topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and induction of the long noncoding RNA Gas5, leading to c-Myc inhibition. PMID: 29158265
- CCND1 , C-MYC , and FGFR1 amplifications were observed in 34.28%, 28.57%, and 17.14% of the 35 samples (invasive ductal breast carcinoma). PMID: 30119151
- Data suggest that MYC induction of REV-ERBalpha is both persistent and recurrent across many inducible MYC model systems. PMID: 28332504
- HUWE1 overexpression could functionally suppress prostate carcinoma development both in vitro and in vivo, possibly by inverse regulation of c-Myc. PMID: 29966975
- Menin functions as an oncogenic regulatory factor that is critical for MYC-mediated gene transcription. PMID: 28474697
- High c-myc expression is associated with colorectal cancer. PMID: 30015962
- Melatonin disturbs SUMOylation-mediated crosstalk between c-Myc and nestin via MT1 activation and promotes the sensitivity of paclitaxel in brain cancer stem cells. PMID: 29654697
- FBP1 modulates the sensitivity of pancreatic cancer cells to BET inhibitors by decreasing the expression of c-Myc. These findings highlight FBP1 as a potential therapeutic niche for patient-tailored therapies. PMID: 30201002
- miR135a directly bound to UCA1 and the 3' untranslated region of cmyc, and UCA1 competed with cmyc for miR135a binding. PMID: 30015867
- MYC directly regulates DANCR and plays an important role in cancer cell proliferation. PMID: 29180471
- This review provides support for the hypothesis that the cooperation of c-Myc with transcriptional cofactors mediates c-Myc-induced cellular functions. We present evidence that recently identified cofactors are involved in c-Myc control of survival mechanisms of cancer cells. PMID: 30261904
- 4-chlorobenzoyl berbamine (CBBM) inhibits the JAK2/STAT3 pathway, leading to reduced c-Myc transcription. Collectively, these findings suggest that CBBM could be a promising lead compound for the treatment of c-Myc-driven diffuse large B cell lymphoma. PMID: 30099568
- Results revealed that C-MYC protein is highly expressed in colon cancer tissues, mainly in the cell nucleus, and was identified as a direct target for mir-184. C-MYC appeared to participate in cell cycle regulation and malignant transformation to colon cancer. PMID: 28782841
- MACC1 and c-Myc are highly expressed in serum and tumor tissues of EC patients. Both are correlated with TNM stage, primary infiltration, and lymph node or distal metastasis. PMID: 29984790
- This study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting versus activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity. PMID: 30181285
- The data demonstrated that 10058F4, a cMyc inhibitor, increased the growth inhibition, G0/G1 phase arrest, and apoptosis of the NALM6 and CEM cells as induced by dexamethasone (DXM), a type of GC. PMID: 29749488
- c-MYC/BCL2 protein co-expression is associated with non-germinal center B-cell in Diffuse Large B-Cell Lymphoma. PMID: 29801406
- c-Myc was capable of upregulating HP1gamma by directly binding to the E-box element in the first intron of HP1gamma gene, and the upregulated HP1gamma, in turn, repressed the expression of miR-451a by enhancing H3K9 methylation at the promoter region of miR-451a. PMID: 28967902
- A subset of pancreatic acinar cell carcinomas shows c-MYC alterations including gene amplification and chromosome 8 polysomy. PMID: 29721608
- Expression and Clinical Significance of LC-3 and P62 in Non-small Cell Lung Cancer. PMID: 29945702
- The findings of the current study demonstrate the presence of the IDH1 R132H mutation in primary human glioblastoma cell lines with upregulated HIF-1alpha expression, downregulating c-MYC activity and resulting in a consequential decrease in miR-20a, which is responsible for cell proliferation and resistance to standard temozolomide treatment. PMID: 29625108
- A novel signal circuit of Stat3/Oct-4/c-Myc was identified for regulating stemness-mediated Doxorubicin resistance in triple-negative breast cancer. PMID: 29750424
- MYC amplification and MYC overexpression occurred almost exclusively in secondary cutaneous angiosarcoma in our series. PMID: 29135507
- High c-myc expression is associated with the development of prostate cancer. PMID: 29554906
- Circular RNA hsa_circRNA_103809 promotes lung cancer progression via facilitating ZNF121-dependent MYC expression by sequestering miR-4302. PMID: 29698681
- Authors conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients. PMID: 29181861
- PCYT1A was upregulated by MYC, which resulted in the induction of aberrant choline metabolism and the inhibition of B-lymphoma cell necroptosis. PMID: 28686226
- Cryptic t(3;8)(q27;q24) and/or MYC-BCL6 linkage associated with MYC expression by immunohistochemistry is frequent in multiple-hit B-cell lymphomas. PMID: 28665415
- CD30+ diffuse large B-cell lymphoma has characteristic clinicopathological features mutually exclusive with MYC gene rearrangement and negatively associated with BCL2 protein expression. PMID: 29666157
- High MYC amplification is associated with HER2 positive breast cancers in African American women. PMID: 29523126
- These data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression via its interaction with MIZ1. PMID: 27339797
- In our study, the c-myc oncogene was amplified in 11.1% of BPH samples. Bivariate analysis failed to reveal any significant association between oncogene amplification and the clinicopathologic variables examined. PMID: 29234244
- Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF1A and HIF1B binding to a MYC enhancer. PMID: 27774982
- Data indicate that miR-34a enhanced the sensitivity to cisplatin by upregulation of c-Myc and Bim pathway. PMID: 29060932
- Luciferase reporter assay showed that c-Myc, an oncogene that regulates cell survival, angiogenesis and metastasis, was a direct target of miR-376a. Over-expression of miR-376a decreased the mRNA and protein levels of c-Myc in A549 cells. PMID: 28741879
- The present findings show that expression of c-MYC has prognostic value in squamous cell carcinoma of the tongue, and could be useful in the choice of therapy. PMID: 28393404
- Multivariable analysis indicated that IPI (P = 0.002), chemotherapy regimens (P = 0.017), and MYC gene rearrangements (P = 0.004) were independent adverse prognostic factors for all diffuse large B cell Lymphoma(DLBCL) patients in this study. Results demonstrated that the poor survival of DLBCL patients with HBV infection was closely involved in chemotherapy regimens, IPI, and MYC gene rearrangements. PMID: 29209623
- MYC extra copy in diffuse large B-cell lymphoma is an independent poor prognostic factor. PMID: 28776574
- The c-Myc/miR-200b/PRDX2 loop regulates colorectal cancer (CRC) progression, and its disruption enhances tumor metastasis and chemotherapeutic resistance in CRC. PMID: 29258530
Q&A
What is the biological significance of MYC phosphorylation at S62?
Phosphorylation of MYC at serine 62 (S62) plays a critical role in modulating its transcriptional activity and stability. This post-translational modification serves as a regulatory mechanism that influences MYC's ability to control gene expression related to cell proliferation, differentiation, and apoptosis. Specifically, phosphorylation at S62 stabilizes the MYC protein by preventing its proteasomal degradation, thereby enhancing its functional lifespan within the cell . Dysregulation of this phosphorylation event has been implicated in oncogenesis, as it can lead to aberrant activation of MYC target genes that promote tumor progression . Additionally, phosphorylation at S62 primes MYC for subsequent phosphorylation at other sites, such as threonine 58 (T58), which is essential for its degradation via ubiquitination pathways .
How is the Phospho-MYC (S62) Recombinant Monoclonal Antibody developed?
The development of the Phospho-MYC (S62) Recombinant Monoclonal Antibody involves several advanced molecular and cellular techniques. Initially, genes encoding antibodies specific to phosphorylated MYC at S62 are isolated from immunized rabbits exposed to synthetic peptides mimicking the phosphorylated human MYC protein . These genes are then cloned into specialized expression vectors and transfected into host suspension cells, such as HEK293 or CHO cells, which are cultured under optimized conditions to express the recombinant antibody . The purification process employs affinity chromatography to isolate the antibody from the culture supernatant, ensuring high specificity and purity. Functional validation is performed using assays like ELISA, Western blotting (WB), and immunofluorescence (IF), confirming the antibody's ability to bind selectively to phosphorylated MYC at S62 .
What experimental applications are suitable for this antibody?
The Phospho-MYC (S62) Recombinant Monoclonal Antibody is validated for a range of experimental applications that include Western blotting (WB), immunohistochemistry-paraffin embedded sections (IHC-P), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) . Each application requires specific optimization of antibody dilution and assay conditions:
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Western Blotting: Recommended dilution ranges from 1:500 to 1:5000 depending on sample type and detection sensitivity .
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IHC-P: Dilution ranges between 1:50 and 1:200 for tissue sections .
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ELISA: Suggested starting concentration is 1 µg/mL, with optimization based on assay requirements .
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Immunofluorescence: Dilutions between 1:20 and 1:200 are recommended for cellular localization studies .
These applications allow researchers to investigate MYC phosphorylation dynamics in various biological contexts, including cancer models.
What controls should be included in experiments using this antibody?
To ensure reliable data interpretation when using the Phospho-MYC (S62) Recombinant Monoclonal Antibody, researchers must include appropriate controls:
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Positive Control: Cell lysates or tissue samples known to express phosphorylated MYC at S62 can serve as positive controls.
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Negative Control: Samples treated with phosphatase enzymes to remove phosphorylation at S62 will confirm antibody specificity.
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Isotype Control: Non-specific rabbit IgG or mouse IgG can be used to rule out non-specific binding.
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Loading Control: Proteins like β-actin or GAPDH should be included in Western blot analyses for normalization purposes .
These controls are critical for validating the specificity and reproducibility of experimental results.
How does phosphorylation at S62 influence MYC degradation pathways?
Phosphorylation at S62 serves as a priming event for subsequent phosphorylation by glycogen synthase kinase-3 beta (GSK3β) at threonine 58 (T58). This stepwise phosphorylation process facilitates MYC's recognition by the SCF(FBXW7) ubiquitin ligase complex, leading to its ubiquitination and proteasomal degradation . Interestingly, dephosphorylation of S62 by protein phosphatase 2A (PPP2CA) destabilizes MYC by promoting its degradation. The interplay between phosphorylation and dephosphorylation events underscores the complexity of MYC regulation within cellular signaling networks .
What are common challenges in detecting phosphorylated MYC using this antibody?
Detecting phosphorylated MYC at S62 can be challenging due to factors such as low endogenous expression levels of phosphorylated MYC in certain cell types or tissues. Additionally, non-specific binding may occur if experimental conditions are not optimized properly. To overcome these challenges:
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Optimize antibody dilution based on preliminary titration experiments.
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Use highly sensitive detection systems such as enhanced chemiluminescence (ECL) for Western blotting.
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Employ stringent washing steps during IHC or IF protocols to reduce background staining.
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Validate specificity using phosphatase-treated samples as negative controls .
Proper optimization ensures accurate detection of phosphorylated MYC.
How can researchers study the functional consequences of S62 phosphorylation in cancer models?
Functional studies on S62 phosphorylation can be conducted using a combination of molecular biology techniques:
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Mutagenesis: Generate site-directed mutants of MYC where serine 62 is replaced with alanine (non-phosphorylatable) or glutamic acid (phosphomimetic) to assess functional outcomes.
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RNA Interference: Knockdown kinases responsible for phosphorylating S62, such as PRKDC or PIM2, using siRNA or shRNA approaches.
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Pharmacological Inhibition: Use inhibitors targeting upstream signaling pathways that regulate S62 phosphorylation.
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Transcriptomic Analysis: Perform RNA sequencing to identify changes in gene expression profiles associated with altered S62 phosphorylation .
These approaches allow researchers to dissect the role of S62 phosphorylation in oncogenic transformation.
What methods are available for quantifying antibody binding affinity?
Quantifying binding affinity is essential for characterizing recombinant monoclonal antibodies like Phospho-MYC (S62). Techniques include:
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Surface Plasmon Resonance (SPR): Measures real-time interactions between the antibody and its target peptide under varying conditions.
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Enzyme-linked Immunosorbent Assay (ELISA): Provides quantitative data on binding efficiency based on absorbance measurements.
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Isothermal Titration Calorimetry (ITC): Evaluates thermodynamic parameters associated with antibody-antigen interactions.
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Flow Cytometry: Quantifies binding affinity in live cells expressing phosphorylated MYC .
Each method offers unique advantages depending on experimental requirements.
