MGAT5 Antibody, Biotin conjugated
Description
Definition and Overview
MGAT5 Antibody, Biotin conjugated is a polyclonal antibody raised against specific epitopes of the MGAT5 enzyme, chemically linked to biotin for enhanced detection in assays like ELISA. MGAT5 (Mannosyl-α-1,6-glycoprotein β-1,6-N-acetyl-glucosaminyltransferase) catalyzes β1-6 GlcNAc branching on N-glycans, influencing cell adhesion, signaling, and cancer progression . The biotin tag enables high-sensitivity detection via streptavidin-based systems.
Development and Specificity
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Immunogen: Synthetic peptide corresponding to amino acids 107–217 of human MGAT5 .
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Conjugate: Biotin, enabling use in streptavidin-HRP or fluorescent systems .
Applications in Research
This antibody is optimized for:
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ELISA: Quantifying MGAT5 expression in serum or cell lysates .
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Functional Studies: Investigating MGAT5’s role in glycosylation-dependent processes like PD-L1/PD-1 interaction .
Key Roles of MGAT5 in Disease
The biotin-conjugated antibody has been instrumental in identifying MGAT5’s role in:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
MGAT5-dependent EGFR N-glycosylation enhances the interaction between EGFR and LGALS3, thereby preventing rapid EGFR endocytosis and prolonging EGFR signaling. Similarly, efficient interaction between TGFB1 and its receptor is facilitated by MGAT5. Further, MGAT5 enhances the activation of intracellular signaling pathways triggered by various growth factors, including FGF2, PDGF, IGF, TGFB1, and EGF. MGAT5-dependent CDH2 N-glycosylation inhibits CDH2-mediated homotypic cell-cell adhesion, contributing to the regulation of downstream signaling pathways and promoting cell migration.
MGAT5's impact extends to the inflammatory response. MGAT5-dependent TCR N-glycosylation enhances the interaction between TCR and LGALS3, limiting agonist-induced TCR clustering and dampening TCR-mediated responses to antigens. This process is essential for normal leukocyte evasation and accumulation at inflammatory sites. MGAT5 inhibits the attachment of monocytes to the vascular endothelium and subsequent monocyte diapedesis. Concurrently, it promotes the proliferation of umbilical vein endothelial cells and angiogenesis, partially mediated by promoting the release of the growth factor FGF2 from the extracellular matrix.
- The best homology model aligns with existing experimental data. The three-dimensional model, the structure of the enzyme catalytic site, and binding information for the donor and acceptor provide valuable insights for studying the catalytic mechanism and designing inhibitors of GnT-V. PMID: 26821880
- Research suggests that oxidative stress induces MGAT5 overexpression through the regulation of the focal adhesion kinase-extracellular signal-regulated kinase signaling pathway. This, in turn, affects the function of endothelial cells, potentially contributing to the pathogenesis of preeclampsia. PMID: 27334383
- PTPalpha has been identified as a novel substrate of N-Acetylglucosaminyltransferase V (GnT-V), suggesting its role in promoting migration in breast cancer cells. PMID: 27965091
- Tunicamycin, an inhibitor of N-glycan biosynthesis, has been shown to enhance the radiosensitivity of U251 cells. These findings suggest that targeting N-linked beta1,6-GlcNAc branches, encoded by N-acetylglucosaminyltransferase V, might hold promise as a therapeutic strategy for glioblastoma treatment. PMID: 26526581
- Knockdown of GnTV significantly suppressed proliferation, migration, and invasion (P<0.05) of SMMC7721/R cells. PMID: 26531171
- GnT-V plays a role in inhibiting trophoblast cell invasion and migration during early pregnancy, potentially through direct or indirect regulation of MMP2/9 activity. PMID: 26349781
- Knockdown of GnT-V using siRNA abolished the level of TGFBR1 and early osteogenic differentiation in DPSCs. This indicates that GnT-V plays a crucial role in hexosamine-induced activation of TGF-b signaling and osteogenic differentiation. PMID: 26583147
- Mgat5 appears to play a significant role in early spontaneous miscarriage in humans. PMID: 26109616
- GnT-V has been shown to accelerate tumor growth. PMID: 26293457
- High expression of GnT-V has been observed in infiltrating cells in skin sections from patients with systemic and localized scleroderma. PMID: 25876794
- MGAT5 protein and gene expression have been studied in uveal and cutaneous melanoma cells. PMID: 26098720
- Human Mgat5 increases amino acid uptake, intracellular levels of glycolytic and TCA intermediates, and HEK293 cell growth. PMID: 25395405
- Research indicates that a deficiency in branched N-glycosylation on TCR, due to reduced MGAT5 gene expression, is a new molecular mechanism underlying ulcerative colitis pathogenesis. PMID: 24334766
- Mgat5-mediated beta-1-6-GlcNAc branched N-glycosylation and subsequent activation of EGFR have been identified as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells. PMID: 23811795
- These findings provide insights into the underlying molecular mechanisms of GnT-V regulation in gastric cancer, with potential translational clinical applications. PMID: 24399258
- GnT-V plays a significant role in metastasis and invasion in gastric cancer cells. PMID: 23563846
- The combination of intratumoral MGAT5 expression and TNM or Kiel staging systems has demonstrated better predictive power for overall survival. PMID: 23107376
- GnT-V directs cancer progression by modulating MMPs (matrix metalloproteinases) in cancer. PMID: 23357422
- Research has shown that the MGAT5 intronic variants rs4953911 and rs3814022 correlate with lower N-glycan branching, reduced surface CTLA-4 in human CD4 + T cell blasts, and associate with multiple sclerosis. PMID: 23351704
- Data demonstrate that knockdown of CD147 inhibited MMP-2 activity of GnT-V-overexpressing cells, indicating that aberrant beta1,6-branches on CD147 are crucial for the induction of MMPs in SMMC-7721 cells. PMID: 23005037
- Findings suggest that GnT-V may be a potential target for predicting nasopharyngeal carcinoma response to radiotherapy. PMID: 22780953
- Research indicates that GnT-V expression is positively related to malignancy in human hepatocellular carcinoma (HCC), suggesting it may be both a differentiation marker and a potential target for the treatment of HCC. PMID: 22537550
- A study identified 33 directly measured and 13 derived glycosylation traits in 3533 individuals. Three novel gene associations (MGAT5, B3GAT1, and SLC9A9) were identified using an additional European cohort. PMID: 21908519
- Inhibiting the expression of N-acetylglucosaminyltransferase V has been shown to inhibit the proliferation of PC-3 cells. PMID: 20584650
- GnT-V expression has been shown to be positively correlated with malignancy in nasopharyngeal carcinoma cells. PMID: 21676538
- Findings suggest that Mgat5 may play an important role during oncogenesis, identifying a potential therapeutic target for pulmonary adenocarcinoma. PMID: 21631992
- The rs1257169(G) allele of MGAT5 has been associated with lower disease severity in multiple sclerosis. PMID: 21115203
- GnT-V was fully active without exogenous cations and in the presence of EDTA. The pH optimum for GnT-V was found to be in the range of 6.5-7.0. PMID: 19846580
- Research suggests that GnT-V could decrease human hepatoma SMMC-7721 cell adhesion and promote cell proliferation partially through RPTPkappa. PMID: 19911372
- The prometastatic effect of N-acetylglucosaminyltransferase V is attributed to the modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching. PMID: 11864986
- A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation, highlighting a novel function of GnT-V distinct from its original glycosyltransferase activity. PMID: 11872751
- Low GnT-V expression is associated with shorter survival and poor prognosis in pStage I overall Non-small cell lung cancer. PMID: 15014031
- Data describe the effect of N-acetylglucosaminyltransferase V on the expressions of other glycosyltransferases involved in the synthesis of surface sialyl Lewis X antigen. PMID: 15044007
- GlcNAc-transferase-V activity is up-regulated in rheumatoid arthritis (RA) and Vitamin D(3)-treated hepatoma cell lines. PMID: 15313475
- GnT-V potentially contributes to placentation in the early phase of pregnancy, possibly regulating the invasion process of trophoblast cells. PMID: 15809094
- Research supports the mechanism that blocking GnT-V expression impairs chaperone and N-glycan-synthesizing enzyme functions, leading to unfolded protein response (UPR) in vivo. PMID: 16467879
- GnT-V is closely related to low malignant potential and good prognosis in patients with bladder cancer. PMID: 16638859
- Glycosylation caused by GnT-V directs integrin beta1 stability and increased delivery to the plasma membrane, subsequently promoting fibronectin-based cell migration and invasion. PMID: 16924681
- Glycosylation changes and a decrease in the transport activity of GLUT1 could be a possible mechanism of endoplasmic reticulum stress induced by down-regulating GnT-V. GnT-V may contribute to the regulation of glucose uptake by modifying the glycosylation of GLUT1 in certain tumor cells. PMID: 17451637
- The study investigated mRNA levels of glycosyltransferases, particularly GnT-V, and found that its expression was decreased in human leukemic cell lines resistant to Epirubicin and Mitoxantrone. PMID: 17488527
- Tissue inhibitor of metalloproteinase-1 (TIMP-1) was identified as a target protein for GnT-V in human colon cancer cell WiDr. PMID: 17878270
- Research suggests that high GnT-V expression was correlated with an unfavorable clinical outcome. PMID: 17971775
- Knock-down of MGAT5 in PC-3 cells attenuated the metastatic ability of prostate cancer cells, as determined by in vitro invasion assays and xenograft animal studies. PMID: 18649738
- GnT-V expression is correlated with a poor prognosis in gastric cancer patients due to metastases. PMID: 18931531
- Decreased GnT-Va activity, resulting from siRNA expression in human carcinoma cells, inhibits ligand-induced EGFR internalization, consequently leading to delayed downstream signal transduction and inhibition of EGF-induced, invasiveness-related phenotypes. PMID: 19225046
- Overexpression of GnT-V in a hepatoma cell line not only induced the addition of beta1,6 GlcNAc branch to the N-glycan of RPTPkappa but also decreased the protein level of RPTPkappa. PMID: 19236842
- High expression of N-acetylglucosaminyltransferase V is associated with mucinous tumors of the ovary. PMID: 19787216
Q&A
What is MGAT5 and what is its biological function?
MGAT5 (mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase) is an enzyme that plays a crucial role in N-glycan biosynthesis in the secretory pathway of human cells. It catalyzes the transfer of N-acetylglucosamine (GlcNAc) to form complex branched N-glycan structures. MGAT5 primes the biosynthesis of N-glycan antenna that has been found to be heavily upregulated in cancer . Correct elaboration of N-linked glycans is essential for normal physiology, and MGAT5-mediated glycosylation significantly influences cellular functions including immune regulation and cancer progression .
What applications is the MGAT5 Antibody, Biotin Conjugated suitable for?
The biotin-conjugated MGAT5 polyclonal antibody (e.g., bs-5841R-Biotin) has been validated for multiple research applications including:
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Western blot (WB)
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Enzyme-linked immunosorbent assay (ELISA)
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Immunohistochemistry with paraffin-embedded sections (IHC-P)
This versatility makes it suitable for multiple experimental approaches in glycobiology and cancer research.
What species reactivity does the MGAT5 Antibody, Biotin Conjugated demonstrate?
The antibody is derived from KLH-conjugated synthetic peptides from human MGAT5 (typically from the immunogen range 201-300/741). It demonstrates confirmed reactivity with human MGAT5 and is predicted to cross-react with MGAT5 from mouse, rat, dog, chicken, and rabbit models . This cross-reactivity makes it valuable for comparative studies across different species, though validation in each new species is recommended.
What are the recommended storage and handling conditions for MGAT5 Antibody?
For optimal performance of biotin-conjugated MGAT5 antibody:
What controls should be included when using MGAT5 Antibody in experiments?
Rigorous experimental design requires appropriate controls:
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Positive control: Cell lines known to express MGAT5, such as AGS and HGC27 gastric cancer cell lines transfected with pcDNA3.1-MGAT5 plasmid .
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Negative control: Include samples where the primary antibody is omitted but all other steps are identical .
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MGAT5 knockout control: Where possible, include MGAT5-deficient cells (created via CRISPR) to confirm antibody specificity .
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Activity verification: Phaseolus vulgaris lectin L (PHA-L) can be used as a control to confirm MGAT5 activity, as it specifically binds MGAT5-mediated N-glycans .
How should researchers optimize IHC protocols for MGAT5 detection?
When performing IHC with biotin-conjugated MGAT5 antibodies:
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Antigen retrieval optimization: Test both heat-induced epitope retrieval and enzymatic retrieval to determine which better exposes the MGAT5 epitope.
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Antibody concentration: Starting with 1μg/μl as provided, titrate to determine optimal signal-to-noise ratio.
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Endogenous biotin blocking: Critical for biotin-conjugated antibodies to prevent false positives.
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Scoring system: Implement the semiquantitative immunoreactivity scoring (IRS) system:
How can MGAT5 Antibody be used to investigate cancer biology?
MGAT5 expression and its glycosylation activity have significant implications in cancer research:
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Expression analysis: MGAT5 expression is significantly higher in various cancer tissues compared to adjacent non-tumor tissues, as demonstrated in head and neck squamous cell carcinoma (HNSCC) . The biotin-conjugated antibody enables precise localization and quantification of MGAT5 in tissue samples.
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Tumor microenvironment studies: MGAT5 expression has been shown to protect tumor cells from cytotoxic T lymphocyte (CTL) killing, suggesting an important role in immune evasion .
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Functional analysis: MGAT5 is required for tumor growth in vivo but not in vitro, indicating context-dependent functions that can be explored using antibody-based detection methods in different experimental models .
What is the role of MGAT5 in relation to immune checkpoint regulation?
Recent research has revealed a critical connection between MGAT5 and immune checkpoint pathways:
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PD-L1 glycosylation: PD-L1 has been identified as a substrate of MGAT5, with N35 and N200 being the primary sites carrying complex N-glycans in HNSCC cells .
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Immunotherapy response prediction: Patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors, suggesting potential biomarker applications .
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Mechanistic studies: The MGAT5 antibody can be used to investigate how branched N-glycans on PD-L1 modulate interaction with the immune checkpoint receptor PD-1, potentially informing new therapeutic approaches .
How can MGAT5 Antibody be integrated into glycoproteomic workflows?
Biotin-conjugated MGAT5 antibodies can enhance glycoproteomic studies:
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Substrate identification: Mass spectrometry (MS)-based glycoproteomic approaches have identified 163 potential protein substrates of MGAT5 . The antibody can be used to validate these candidates through immunoprecipitation and western blotting.
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Functional analysis: Substrate proteins of MGAT5 have been found to regulate pathways related to T cell proliferation and activation, which can be further investigated using antibody-based approaches .
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Glycan structure characterization: Combined with lectin affinity methods (particularly PHA-L), the antibody can help characterize MGAT5-specific glycosylation patterns in various physiological and pathological contexts.
How can researchers address weak or non-specific staining with MGAT5 Antibody?
When encountering issues with MGAT5 antibody staining:
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For weak signal:
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Increase antibody concentration or incubation time
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Optimize antigen retrieval conditions
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Consider signal amplification systems compatible with biotin conjugates
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For non-specific binding:
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Validate antibody specificity by Western blot analysis before IHC applications
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Increase blocking time using both protein and biotin-specific blocking reagents
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Include additional washing steps with detergents
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Antibody validation: Prior to extensive experimentation, verify antibody specificity using Western blot analysis in control and MGAT5-overexpressing cell lines .
What are the challenges in quantifying MGAT5 expression levels?
Accurate quantification requires consideration of several factors:
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IHC analysis: The semiquantitative IRS system combining staining intensity and percentage of positive cells offers a standardized approach .
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Western blot quantification: Densitometric analysis normalized to appropriate loading controls with careful validation of linearity range.
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Heterogeneity considerations: MGAT5 expression may vary within tumors, requiring multiple sampling and careful interpretation of whole-tissue expression data.
How should researchers interpret MGAT5 expression in relation to glycosylation status?
Interpretation requires integrating multiple data points:
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Enzyme expression vs. activity: MGAT5 protein detection does not always correlate directly with enzymatic activity; complementary approaches using PHA-L lectin can confirm functional activity .
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Context-dependent interpretation: The significance of MGAT5 expression differs between in vitro and in vivo settings, with MGAT5-deficient cells showing no growth deficiency in vitro despite its requirement for tumor growth in vivo .
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Correlation with clinical outcomes: Analysis should include correlation with patient demographics, tumor stage, and treatment response, particularly in relation to immunotherapy .
What emerging applications exist for MGAT5 Antibody in precision medicine?
The biotin-conjugated MGAT5 antibody has potential applications in emerging precision medicine approaches:
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Immunotherapy response prediction: MGAT5 shows promise as a biomarker to predict patients' responses to anti-PD-1 therapy, which could be developed into a companion diagnostic test .
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Targeted therapeutic development: Understanding MGAT5's role in modifying PD-L1 and other immune-related glycoproteins could lead to novel therapeutic targets focused on specific glycosylation sites (N35 and N200 of PD-L1) .
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Combinatorial biomarker panels: Integrating MGAT5 detection with other glycosylation markers may provide more comprehensive predictive value for treatment selection.
How might engineered MGAT5 variants be detected using modified antibodies?
Recent protein engineering approaches have yielded modified MGAT5 variants:
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Bioorthogonal substrate analogs: Engineered MGAT5 variants (such as BH-MGAT5) that preferentially use bioorthogonal substrate analogs (like UDP-GlcNButAz) over native UDP-GlcNAc have been developed .
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Detection challenges: Modified antibodies may be needed to specifically recognize these engineered variants in experimental settings.
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Structural considerations: The crystal structure of MGAT5 provides insights for engineering-specific detection reagents that can distinguish between wild-type and modified variants .
What considerations are important for multiplexed detection of MGAT5 with other glycosylation markers?
Future research will benefit from multiplexed approaches:
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Compatible detection systems: When using biotin-conjugated MGAT5 antibody, other detection reagents should employ non-biotin systems to avoid cross-reactivity.
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Sequential staining protocols: For tissue sections, carefully optimized sequential staining with other glycosylation markers will provide spatial context.
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Multi-omic integration: Combining antibody-based MGAT5 detection with glycan profiling, transcriptomics, and proteomics will provide the most comprehensive understanding of glycosylation biology in health and disease.
