MAN2A1 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- QSAR models utilizing fragmented QM-DFT descriptors offer valuable insights in structure-based drug design, especially when empirical and force field methods reach their limits and quantum mechanics effects significantly influence ligand-receptor interactions. These optimized models can be applied in lead optimization processes for developing therapeutics targeting MAN2A1. PMID: 27035259
- Many human tumor types and cancer cell lines express the MAN2A1-FER fusion, which contributes to increased proliferation and invasiveness of cancer cells. In mice, this fusion has been shown to exhibit liver oncogenic activity. PMID: 28245430
- Inhibition of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells induces a unique type of hybrid glycosylation containing a partially processed mannose moiety. PMID: 17466984
- Research involving the deletion of the MAN2A1 homologue in mice has led to the development of a systemic autoimmune disease resembling human systemic lupus erythematosus. PMID: 11158608
Q&A
What is MAN2A1 and what is its biological significance?
MAN2A1 (Mannosidase Alpha Class 2A Member 1) is an enzyme involved in N-glycan maturation that plays a critical role in protein glycosylation processes. It functions in the Golgi apparatus where it cleaves specific mannose residues during N-glycan processing. MAN2A1 has emerged as a key immunomodulatory gene with significant implications in cancer biology .
N-linked glycosylation is a critical regulator in the tumor microenvironment that can be manipulated to enhance antitumor immunity . Research has shown that MAN2A1 is downregulated in colorectal cancer tissues compared to adjacent normal tissues, and further downregulated in metastatic colorectal cancer compared to non-metastatic cases . This suggests MAN2A1 may function as a tumor suppressor in certain contexts.
What detection methods are recommended for MAN2A1 expression analysis?
Several validated methods exist for detecting MAN2A1 expression:
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Western Blotting: Anti-mouse MAN2A1 Antibody (Santa Cruz Biotechnology, #sc-376909) and anti-human MAN2A1 antibody (Santa Cruz Biotechnology, #sc-377204) have been successfully used in published research . Standard protein extraction protocols followed by SDS-PAGE separation are effective, with GAPDH, vinculin, or ERK2 serving as reliable loading controls.
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RT-PCR: Real-time polymerase chain reaction provides sensitive quantification of MAN2A1 mRNA expression. This approach has been validated in colorectal cancer studies comparing tumor tissues with adjacent normal tissues .
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Immunohistochemistry: For tissue section analysis, optimized protocols typically include antigen retrieval, primary antibody incubation (typically at 1:100-1:200 dilution), and visualization with appropriate secondary antibody systems.
How should researchers interpret varying MAN2A1 expression across tissue types?
Interpreting MAN2A1 expression requires careful consideration of tissue context:
How should researchers design experiments to study MAN2A1 functional effects?
When designing experiments to investigate MAN2A1 function:
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Gene Knockout Approaches: CRISPR-Cas9 systems have been effectively used for MAN2A1 knockout. Specific gRNA sequences targeting MAN2A1 can be cloned into lentiviral vectors (e.g., PLKO3G-GFP vector) for stable knockout cell line generation .
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Verification of Knockout Efficiency: Western blotting using anti-MAN2A1 antibodies should be performed to confirm protein elimination. Additional functional validation may include altered glycosylation pattern analysis using lectin binding assays.
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Phenotypic Assessment: For cancer-related research, cell proliferation, migration, invasion assays, and in vivo tumor growth models have proven informative. The mixing of MAN2A1-knockout (GFP-positive) cells with parental (GFP-negative) cells at a 1:1 ratio provides an elegant competition assay to assess relative fitness .
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Pharmacological Inhibition: Swainsonine, a MAN2A1 inhibitor, can be used as a complementary approach to genetic knockdown experiments, particularly when investigating the therapeutic potential .
What are the optimal conditions for MAN2A1 antibody use in Western blotting?
For optimal Western blotting results with MAN2A1 antibodies:
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Sample Preparation: Total protein extraction from cultured cells or tissue samples should use RIPA buffer supplemented with protease inhibitors. For glycoproteins like MAN2A1, avoid excessive heating which may cause aggregation.
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Antibody Selection and Dilution:
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Secondary Antibody Detection: Goat anti-rabbit secondary antibody (Cell Signaling Technology, #7074s) or Goat anti-mouse secondary antibody (Cell Signaling Technology, #7076s) have been successfully employed .
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Visualization System: Standard ECL detection systems, such as the CLINX imaging system, provide adequate sensitivity for MAN2A1 detection .
How does MAN2A1 inhibition enhance immune checkpoint blockade efficacy?
The mechanism by which MAN2A1 inhibition enhances immune checkpoint blockade involves several steps:
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Enhanced Cancer Cell Susceptibility: Loss of Man2a1 in cancer cells significantly increases their sensitivity to T-cell-mediated killing. This suggests altered glycosylation patterns affect cancer cell recognition by immune effectors .
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Improved Tumor Infiltration: Man2a1 knockout facilitates higher cytotoxic T-cell infiltration in tumors under anti-PD-L1 treatment, improving immune surveillance .
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Synergistic Effects: Pharmacologic inhibition of MAN2A1 with swainsonine synergizes with anti-PD-L1 treatment in multiple cancer models (melanoma and lung cancer). This combination therapy shows efficacy where each treatment alone has limited effect .
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PD-L1 Glycosylation Effects: PD-L1 is highly glycosylated, and N-glycosylation stabilizes PD-L1 protein. MAN2A1 inhibition likely alters PD-L1 glycosylation patterns, potentially affecting its stability and interaction with PD-1 .
What molecular mechanisms link MAN2A1 to cancer progression and metastasis?
The mechanisms connecting MAN2A1 to cancer progression involve multiple pathways:
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Signaling Pathway Involvement: Gene Set Enrichment Analysis (GSEA) has identified that MAN2A1 expression affects several cancer-related pathways, including:
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Lymph Node Metastasis Correlation: Logistic regression analysis has demonstrated that MAN2A1 expression is significantly associated with lymph node metastasis status (OR: 0.725, 95% CI: 0.526–1.001, P=0.05) .
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TNM Stage Association: Lower MAN2A1 expression correlates with advanced TNM stages (stages III and IV vs. stages I and II, OR: 0.684, 95% CI: 0.494–0.947, P=0.022) .
What are the technical challenges when using MAN2A1 antibodies for immunohistochemistry?
When performing immunohistochemistry with MAN2A1 antibodies, researchers should consider:
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Fixation Optimization: Overfixation can mask MAN2A1 epitopes. For formalin-fixed paraffin-embedded tissues, fixation time should be standardized (typically 12-24 hours in 10% neutral buffered formalin).
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Antigen Retrieval Methods: Heat-induced epitope retrieval using citrate buffer (pH 6.0) typically provides optimal results for MAN2A1 detection, though EDTA-based buffers (pH 9.0) may be tested as alternatives.
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Antibody Validation Controls: Include known positive tissues (e.g., normal colon) and negative controls (primary antibody omission) in each staining run. For colorectal cancer studies, adjacent normal tissue provides an excellent internal control.
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Signal Amplification Strategies: For low-expressing samples, consider tyramide signal amplification or polymer-based detection systems to enhance sensitivity while maintaining specificity.
How should researchers reconcile contradictory findings regarding MAN2A1's role in different cancer types?
When addressing discrepancies in MAN2A1 research:
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Cancer Type Specificity: MAN2A1's role may vary by cancer type. While it shows tumor suppressor characteristics in colorectal cancer , its function may differ in other malignancies.
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Context-Dependent Effects: Consider the microenvironmental context, including immune infiltration status, which may modulate MAN2A1's impact.
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Technical Variations: Different antibodies, detection methods, and scoring systems across studies may contribute to apparently contradictory results.
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Genetic Background Influence: Host genetic factors may modify MAN2A1's effects, suggesting the importance of characterizing genetic backgrounds in experimental models.
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Fusion Protein Considerations: MAN2A1-FER fusion has been reported in some cancers, which functions differently from wild-type MAN2A1. Always confirm which form is being detected .
What is the relationship between MAN2A1 expression and patient survival in cancer studies?
The prognostic value of MAN2A1 has been systematically investigated:
The table below summarizes the clinical correlations:
| Clinical Variable | Low MAN2A1 | High MAN2A1 | P-value |
|---|---|---|---|
| TNM Stage III-IV | 157 | 110 | 0.02 |
| Lymph Node Metastasis | 153 | 111 | 0.05 |
| Distant Metastasis | 56 | 32 | 0.056 |
How might MAN2A1-targeted therapies be developed for cancer treatment?
Potential approaches for MAN2A1-targeted therapeutic development include:
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Inhibitor Optimization: While swainsonine has shown efficacy in preclinical models, especially in combination with anti-PD-L1 therapy, more selective MAN2A1 inhibitors with improved pharmacokinetic properties could be developed .
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Combination Therapy Strategies: Based on synergistic effects with immune checkpoint inhibitors, rational combination approaches could be explored with other immunotherapy modalities.
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Biomarker-Guided Treatment: MAN2A1 expression levels could serve as a predictive biomarker for patient stratification in immunotherapy trials.
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Glycoengineering Approaches: Beyond direct inhibition, modulating specific glycosylation patterns affected by MAN2A1 might provide alternative therapeutic strategies.
What advanced research techniques are emerging for studying MAN2A1 glycosylation functions?
Cutting-edge techniques for investigating MAN2A1 function include:
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Glycoproteomics: Mass spectrometry-based approaches can identify specific N-glycan structures affected by MAN2A1 modulation, providing insight into functional consequences.
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CRISPR Activation/Interference: CRISPRa and CRISPRi systems allow for precise modulation of MAN2A1 expression levels without complete knockout, enabling dose-response studies.
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Patient-Derived Organoids: Three-dimensional organoid cultures from patient tumors provide physiologically relevant systems to study MAN2A1 function in personalized contexts.
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Single-Cell Glycomics: Emerging technologies for single-cell analysis of glycosylation patterns can reveal heterogeneity in MAN2A1 activity within tumor microenvironments.
