MBL2 Antibody

What is MBL2 and what is its biological significance?

Mannose-binding lectin 2 (MBL2) is a calcium-dependent lectin involved in innate immune defense. The protein plays a crucial role in recognizing pathogen-associated molecular patterns (PAMPs) by binding to mannose, fucose, and N-acetylglucosamine on various microorganisms, which activates the lectin complement pathway. MBL2 also facilitates the clearance of apoptotic cells by binding to late apoptotic cells and necrotic cells (but not early apoptotic cells), thereby enhancing their uptake by macrophages. Additionally, MBL2 may bind DNA .

The human MBL2 protein has a calculated molecular weight of approximately 26 kDa, though it is typically observed at around 32 kDa in Western blots due to post-translational modifications . It is primarily produced in the liver and features two important structural domains: the C-terminal carbohydrate recognition domain (CRD) and the N-terminal collagen domain .

What are the common applications for MBL2 antibodies in research?

MBL2 antibodies are utilized across multiple experimental platforms to investigate its role in innate immunity and disease pathogenesis. Common applications include:

Researchers should note that application-specific optimization is recommended as sample types and experimental conditions can significantly affect performance .

How should antigen retrieval be optimized for MBL2 immunohistochemistry?

Effective antigen retrieval is critical for accurate MBL2 detection in tissue samples. Based on validated protocols, two primary methods are recommended:

• TE Buffer Method (Preferred): Use Tris-EDTA buffer at pH 9.0 for heat-induced epitope retrieval. This alkaline pH has shown superior results for many MBL2 antibodies .

• Citrate Buffer Alternative: Some antibodies perform adequately with citrate buffer at pH 6.0 . This should be considered as a secondary option if the TE buffer method yields suboptimal results.

The heat-induced epitope retrieval process should be performed thoroughly, typically using pressure cookers or microwave methods. For paraffin-embedded tissues, a complete deparaffinization and rehydration process should precede antigen retrieval. When staining liver tissue (a major site of MBL2 production), particular attention should be paid to reducing background staining through adequate blocking steps .

What are the recommended protocols for Western blot detection of MBL2?

For optimal Western blot detection of MBL2, researchers should follow these methodological considerations:

• Sample Preparation:

  • For serum samples: Dilute 1:10 to 1:50 in loading buffer

  • For tissue extracts: Conventional protein extraction with protease inhibitors is essential

  • Blood tissue samples have shown consistent MBL2 detection

• Gel Selection and Transfer:

  • 10-12% SDS-PAGE gels are typically suitable

  • Transfer to PVDF membranes (rather than nitrocellulose) may improve detection

  • Expected molecular weight: While the calculated weight is 26 kDa, MBL2 is typically observed at 32 kDa

• Antibody Dilution and Incubation:

  • Primary antibody: 1:500-1:2000 dilution with overnight incubation at 4°C is recommended

  • Secondary antibody: HRP-conjugated anti-species antibody at 1:5000-1:10000

• Detection Systems:

  • Enhanced chemiluminescence (ECL) detection systems are suitable

  • For low expression samples, more sensitive ECL substrates may be necessary

When troubleshooting, researchers should note that MBL2 migrates differently than its predicted molecular weight, and validation with positive controls (such as human blood tissue) is strongly recommended .

How do polymorphisms in the MBL2 gene affect protein function and disease susceptibility?

MBL2 gene polymorphisms, particularly in exon 1, significantly impact serum MBL levels and subsequent disease susceptibility. Research has identified several key polymorphic alleles:

*Frequencies based on study of 122 patients with acute lymphoid leukemia (ALL)

Methodologically, researchers investigating MBL2 polymorphisms should combine genotyping (typically through PCR and sequencing) with quantitative serum MBL level measurements to establish genotype-phenotype correlations in their specific study populations.

What is the relationship between MBL2 expression and hepatocellular carcinoma (HCC)?

Recent multi-omics analyses and experimental validation have revealed a significant role for MBL2 in hepatocellular carcinoma (HCC) pathogenesis and progression. Key findings include:

• Expression and Prognosis:

  • Lower expression of MBL2 in HCC patients correlates with unfavorable prognosis

  • Bioinformatics analyses show close relationships among MBL2 downregulation, tumor-associated proliferation/metastasis pathways, and immunosuppressive microenvironments

• Functional Effects:

  • Overexpression of MBL2 directly inhibits the proliferation and metastasis of HCC cells

  • Experimental validation using cell counting kit-8 assays, colony formation assays, transwell migration assays, and wound healing assays have confirmed this inhibitory effect

• Regulatory Mechanisms:

  • MBL2 expression is regulated by miR-34c-3p, as confirmed by dual-luciferase reporter assays

  • This microRNA represents an upstream mechanism of MBL2 downregulation in HCC

For researchers investigating MBL2 in HCC contexts, methodological approaches should include:

• Comprehensive expression analysis across HCC tissue cohorts

• Correlation of expression with clinical outcomes

• In vitro functional studies using overexpression systems

• Validation in multiple cell lines and potentially in vivo models

• Investigation of regulatory mechanisms through microRNA binding studies

These findings collectively suggest that MBL2 could serve as a potential therapeutic target for HCC, with increasing MBL2 levels representing a promising strategy for treatment .

How can researchers effectively overexpress MBL2 in experimental models?

Based on successful experimental approaches documented in the literature, MBL2 overexpression can be achieved through the following validated methodology:

• Vector Construction:

  • Human MBL2 cDNA should be constructed and packaged into lentiviral vectors

  • Recommended vector backbone: pHBLV-U6-MCS-CMV-ZsGreen-PGK-puromycin

  • Both experimental (LV-MBL2) and negative control (LV-Ctrl) vectors should be prepared

• Transfection Protocol:

  • Perform transfection in a biological safety cabinet following strict biosafety protocols

  • Optimal viral titer should be determined for each cell line

  • For hepatocellular carcinoma cell lines, standard lentiviral transfection protocols have proven effective

• Validation of Overexpression:

  • Confirm successful overexpression through Western blotting

  • qRT-PCR can provide quantitative assessment of increased mRNA levels

  • Functional validation through known MBL2-dependent assays is recommended

• Functional Assessment:

  • Cell proliferation analysis: Cell counting kit-8 assay

  • Migration capability: Transwell migration assay

  • Wound healing dynamics: Wound healing assay at 0 and 24 hours after scratching

Researchers should note that MBL2 overexpression has been successfully used to demonstrate its tumor-suppressive effects in HCC models, with significant inhibition of cell proliferation and metastasis observed .

What criteria should guide selection among different MBL2 antibody options?

When selecting an MBL2 antibody for research applications, several technical factors should be considered:

For critical research, antibodies with extensive validation evidence (ideally multiple applications and citations) should be prioritized. The antibody's target epitope should also align with the research question—for instance, if studying a specific domain's function, select antibodies targeting that region.

What controls are essential for validating MBL2 antibody specificity?

Rigorous validation is essential for ensuring reliable results with MBL2 antibodies. The following controls should be integrated into experimental workflows:

• Positive Controls:

  • Human liver tissue (major site of MBL2 production) for IHC applications

  • Human blood tissue for Western blot applications

  • HEK293T cells transfected with MBL2 expression vectors

• Negative Controls:

  • Nonimmune normal serum or TBS (Tris-buffered saline)

  • Isotype-matched control antibodies

  • Tissues known to lack MBL2 expression

• Specificity Controls:

  • Knockdown/knockout validation: siRNA or CRISPR-mediated depletion of MBL2

  • Peptide competition assays: Pre-incubation with immunogen peptide should abolish signal

  • Multiple antibody validation: Using different antibodies targeting distinct epitopes

• Technical Controls:

  • Secondary antibody-only controls to assess background

  • Endogenous peroxidase blocking for IHC applications

  • Loading controls (such as β-actin) for Western blot normalization

Researchers should document validation results thoroughly, as antibody performance can vary between applications and sample types. For IHC applications specifically, the staining intensity score system (ranging from 0-4 based on percent positivity) provides a quantitative assessment method, with 0 representing 0%, 1 indicating 1–25%, 2 denoting 26–50%, 3 signifying 51–75%, and 4 representing >75% positivity .

How is MBL2 being investigated as a therapeutic target in disease contexts?

Recent research has identified MBL2 as a promising therapeutic target, particularly in oncology and immunology contexts:

• Hepatocellular Carcinoma (HCC):

  • Comprehensive multi-omics analyses have established MBL2 as a key regulator in HCC

  • Lower expression of MBL2 correlates with unfavorable prognosis

  • Experimental evidence confirms that MBL2 overexpression inhibits HCC proliferation and metastasis

  • miR-34c-3p has been identified as a regulatory microRNA that could be targeted therapeutically to modulate MBL2 expression

• Immunodeficiency Contexts:

  • MBL2 polymorphisms significantly impact susceptibility to infections, particularly in immunocompromised patients

  • In acute lymphoid leukemia (ALL) patients, specific genotypes (particularly O/O) show increased susceptibility to viral infections

  • These findings suggest potential for replacement therapy or immunomodulatory approaches targeting MBL2

• Methodological Approaches for Therapeutic Development:

  • Gene therapy approaches using lentiviral vectors for MBL2 overexpression

  • microRNA inhibitors targeting miR-34c-3p to upregulate MBL2

  • Recombinant MBL2 protein administration for replacement therapy

  • Small molecule modulators of MBL2 expression or function

Researchers focusing on MBL2 as a therapeutic target should consider combination approaches, particularly in oncology contexts where multi-modal therapeutic strategies are typically required. Additionally, patient stratification based on MBL2 genotype could identify subpopulations most likely to benefit from MBL2-targeted interventions .

What are the technical challenges in studying MBL2 interactions with the immune microenvironment?

Investigating MBL2's interactions within the immune microenvironment presents several technical challenges that researchers should address methodologically:

• Microenvironment Complexity:

  • Bioinformatics analyses reveal relationships between MBL2 downregulation and immunosuppressive microenvironments

  • Studying these interactions requires sophisticated multi-parameter approaches

• Technical Approaches and Solutions:

  • Single-cell analysis: To resolve cell-specific MBL2 expression and effects

  • 3D culture systems: To better recapitulate the spatial organization of the tissue microenvironment

  • Multi-parameter flow cytometry: To simultaneously assess MBL2 and immune cell markers

  • Multiplex immunohistochemistry: To visualize MBL2 alongside immune cell populations in tissue contexts

  • Functional immune assays: To determine how MBL2 modulates specific immune functions

• Experimental Design Considerations:

  • Include appropriate controls for each cell type and activation state

  • Account for the context-dependent nature of immune interactions

  • Design experiments that can distinguish direct versus indirect effects of MBL2

  • Consider temporal dynamics, as immune responses evolve over time

• Translational Relevance:

  • Correlate experimental findings with clinical outcomes

  • Develop predictive biomarkers based on MBL2 and immune parameters

  • Consider combination approaches targeting both MBL2 and immune checkpoints

These technical approaches are particularly relevant for oncology research, where understanding the interplay between MBL2, tumor cells, and the immune microenvironment could reveal new therapeutic strategies .