MAN2B2 Antibody

What is MAN2B2 and what cellular functions does it perform?

MAN2B2, also known as alpha-mannosidase 2B2, is an enzyme that plays a crucial role in the glycosylation process within cells, specifically in the breakdown of complex carbohydrates in the lysosome . The protein functions as a lysosomal alpha-mannosidase with specificity for cleaving the α1–6-mannose residue of N-linked glycans, and is particularly important in cleaving the Chitobiase (CTBS) product Man2GlcNac1 to generate Man1GlcNac1 . This enzymatic activity is essential for proper glycoprotein degradation and recycling within the cell, maintaining cellular homeostasis and proper function of numerous physiological processes. Dysregulation of MAN2B2 has been implicated in lysosomal storage disorders and other metabolic diseases, highlighting its importance in normal cellular function and metabolism . Recent research has revealed that MAN2B2 deficiency leads to a specific congenital disorder of glycosylation characterized by immune dysregulation, developmental delay, and multiple organ system involvement .

What applications are MAN2B2 antibodies validated for in research settings?

MAN2B2 antibodies, such as the PACO56250 polyclonal antibody, have been validated for multiple research applications that enable the investigation of MAN2B2 expression and function. The primary validated applications include Western Blotting (WB), which allows for the detection and quantification of MAN2B2 protein in tissue or cell lysates, with recommended dilutions of 1:500-1:5000 . Immunohistochemistry (IHC) is another validated application with recommended dilutions of 1:20-1:200, permitting visualization of MAN2B2 distribution in tissue sections . Additionally, Enzyme-Linked Immunosorbent Assay (ELISA) has been validated with recommended dilutions of 1:2000-1:10000 for quantitative detection of MAN2B2 in solution . These validated applications enable researchers to conduct comprehensive studies on MAN2B2 expression patterns, subcellular localization, and molecular interactions in various experimental models. When selecting an antibody for your specific research needs, it is critical to confirm the validation data for your particular application and experimental conditions.

How specific are commercially available MAN2B2 antibodies for human samples?

Commercial MAN2B2 antibodies, such as the PACO56250 polyclonal antibody, are designed with high specificity for human MAN2B2 protein samples . This specificity is achieved through careful immunogen selection, often using recombinant human MAN2B2 protein fragments as the immunizing antigen. For example, the PACO56250 antibody was developed using a recombinant human epididymis-specific alpha-mannosidase protein fragment (amino acids 347-523) as the immunogen . The antibody undergoes purification processes, including Protein G purification to achieve >95% purity, enhancing its specificity for the target protein . While primarily reactive with human samples, cross-reactivity testing has shown that some MAN2B2 antibodies may detect the protein in rat kidney tissue, suggesting potential utility in comparative studies across species . When planning experiments, researchers should conduct preliminary validation studies to confirm the antibody's specificity in their particular experimental system, especially when working with non-human models or examining specific cell types or tissues not previously tested.

How can MAN2B2 antibodies be used to study congenital disorders of glycosylation?

MAN2B2 antibodies serve as essential tools for investigating the molecular mechanisms underlying MAN2B2-associated congenital disorders of glycosylation (CDG), a rare inborn error of metabolism . In research settings, these antibodies can be used to assess MAN2B2 protein expression levels in patient-derived samples compared to healthy controls using Western blotting techniques, which helps determine if pathogenic variants affect protein stability or expression . Interestingly, research has shown that in some cases of MAN2B2-CDG, the protein level remains unchanged despite functional impairment, suggesting that certain variants affect enzymatic activity rather than protein expression . Immunohistochemistry using MAN2B2 antibodies allows for the visualization of protein localization in affected tissues, potentially revealing abnormal subcellular distribution patterns associated with disease states . Furthermore, these antibodies can be employed in co-immunoprecipitation experiments to identify altered protein-protein interactions in the glycosylation pathway that may contribute to disease pathology . When combined with glycan profiling techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody-based studies provide comprehensive insights into how MAN2B2 variants disrupt N-glycan structures and glycoprotein functions, expanding our understanding of the pathophysiology of these rare disorders .

What are the optimal conditions for using MAN2B2 antibodies in immunohistochemistry?

Optimizing conditions for MAN2B2 antibody use in immunohistochemistry (IHC) requires careful attention to multiple parameters to ensure specific and reproducible staining. Antigen retrieval is a critical first step, with heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) typically showing good results for MAN2B2 detection in formalin-fixed, paraffin-embedded (FFPE) tissues . The optimal antibody dilution range for MAN2B2 antibodies in IHC applications is typically between 1:20 and 1:200, but researchers should perform dilution series experiments to determine the optimal concentration for their specific tissue and fixation conditions . Blocking procedures should include both protein blocking (typically with 5-10% normal serum from the same species as the secondary antibody) and peroxidase blocking (with 0.3-3% hydrogen peroxide) to minimize background staining and false positive results . Incubation conditions affect staining quality, with overnight incubation at 4°C often yielding better signal-to-noise ratios than shorter incubations at room temperature . Detection systems should be selected based on the required sensitivity, with avidin-biotin complex (ABC) or polymer-based systems generally providing good results for MAN2B2 detection . Positive and negative controls are essential for result interpretation, with rat kidney tissue serving as a useful positive control for MAN2B2 antibodies, while omission of primary antibody or use of isotype control antibodies provides appropriate negative controls .

How can MAN2B2 antibodies be used alongside glycan profiling techniques?

Integrating MAN2B2 antibody-based analyses with glycan profiling techniques creates a powerful approach for comprehensive investigation of glycosylation disorders and normal mannosidase function. Researchers can employ a sequential workflow where Western blotting with MAN2B2 antibodies is used to quantify protein expression levels, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess N-glycan profiles and identify specific accumulating glycan structures resulting from enzymatic deficiency . This approach proved valuable in characterizing MAN2B2-CDG, where protein expression remained normal despite dramatic alterations in N-glycan patterns, with marked accumulation of Man2GlcNac2 glycans in patient fibroblasts compared to controls . Immunoprecipitation using MAN2B2 antibodies can isolate the protein for subsequent enzymatic activity assays measuring the conversion of Man2GlcNac1 to Man1GlcNac1, providing direct functional data that complements structural glycan profiling . Cell models can be developed where MAN2B2 expression is manipulated (overexpression, knockdown, or variant introduction) and monitored via antibody-based methods, while corresponding changes in the cellular glycome are tracked through techniques like electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry . For clinical research, correlative studies comparing MAN2B2 protein levels in patient samples (detected by antibodies) with serum N-glycan profiles and urinary mannose-rich oligosaccharides can establish biomarkers for diagnosis and treatment monitoring of MAN2B2-related disorders .

What controls should be included when validating MAN2B2 antibody specificity in experimental systems?

Comprehensive validation of MAN2B2 antibody specificity requires inclusion of multiple control types to ensure reliable and reproducible research outcomes. Positive tissue controls are essential, with rat kidney tissue having been documented to express detectable levels of MAN2B2 and thus serving as an appropriate positive control for antibody validation . Negative controls should include tissues or cell lines known not to express MAN2B2, as well as technical negative controls where the primary antibody is omitted or replaced with non-specific IgG from the same species at equivalent concentration . Genetic knockdown or knockout controls provide the most stringent validation, where MAN2B2 expression is reduced or eliminated using siRNA, shRNA, or CRISPR-Cas9 technology, with subsequent demonstration of corresponding reduction or loss of antibody signal . Peptide competition assays, in which the antibody is pre-incubated with excess immunizing peptide before application to samples, should result in signal abolishment if the antibody is truly specific . Overexpression controls using cell lines transfected with MAN2B2 expression vectors should demonstrate increased signal intensity proportional to expression levels . For studying MAN2B2 variants, isogenic cell lines expressing either wild-type or mutant forms of MAN2B2 serve as valuable controls, as demonstrated in studies where cells transfected with wild-type MAN2B2 were compared to those expressing the p.Asp38Asn variant or compound heterozygous variants (c.384G>T; c.926T>A) .

How do MAN2B2 antibodies contribute to studying immune dysregulation in MAN2B2-CDG?

MAN2B2 antibodies provide crucial insights into the molecular mechanisms underlying immune dysregulation in MAN2B2-CDG by enabling detailed analysis of protein expression patterns in immune cells and tissues. Studies of MAN2B2-CDG patients have revealed distinct immune phenotypes, including inversion of the T-helper/T-cytotoxic (Th/Tc) ratio, increased B-cell numbers, decreased IgG levels, and elevated inflammatory markers, which can be correlated with MAN2B2 protein expression through immunoblotting analysis . Immunohistochemistry using MAN2B2 antibodies enables visualization of protein distribution in lymphoid tissues and identification of cell-specific expression patterns that may explain the selective immune cell abnormalities observed in patients . Flow cytometry applications incorporating permeabilization and intracellular staining with MAN2B2 antibodies allow for quantitative assessment of protein expression across different immune cell subsets, potentially revealing cell type-specific differences in MAN2B2 expression or localization that contribute to immune dysregulation . Research using MAN2B2 antibodies has demonstrated that glycosylation defects resulting from MAN2B2 deficiency affect critical immune regulatory proteins, including SAP30 and TGFB1, which may be core elements in inducing immune dysregulation in MAN2B2-CDG patients . When combined with functional immunological assays, antibody-based detection of MAN2B2 enables correlation between protein expression/function and specific immune abnormalities, providing a foundation for developing targeted therapeutic approaches for the immune manifestations of MAN2B2-CDG .

How can MAN2B2 antibodies aid in developing and validating therapeutic approaches for MAN2B2-CDG?

MAN2B2 antibodies serve as essential tools in the development and validation of therapeutic approaches for MAN2B2-CDG through multiple applications across the drug development pipeline. In preclinical research, these antibodies enable screening of compounds or biological agents that might stabilize mutant MAN2B2 protein or enhance its residual enzymatic activity, with Western blotting providing quantitative assessment of protein levels following treatment . For gene therapy approaches, MAN2B2 antibodies can verify successful transduction and expression of wild-type MAN2B2 in patient-derived cells, as demonstrated in studies where transduction of wild-type MAN2B2 into patient fibroblasts normalized the N-linked glycan profile . Pharmacodynamic biomarker development is facilitated by MAN2B2 antibodies, which can measure changes in protein expression or localization in accessible patient samples (such as peripheral blood cells or skin fibroblasts) in response to therapeutic interventions . In enzyme replacement therapy development, these antibodies are crucial for confirming the integrity and stability of recombinant MAN2B2 protein formulations before administration, as well as tracking their uptake and distribution in target tissues . Patient stratification for clinical trials may be improved by using MAN2B2 antibodies to classify individuals based on residual protein expression levels or subcellular localization patterns, potentially identifying subgroups more likely to respond to specific therapeutic approaches . For combination therapy approaches targeting both MAN2B2 and immune dysregulation, antibody-based monitoring of MAN2B2 expression alongside immune parameters provides comprehensive assessment of treatment efficacy across multiple disease manifestations .

How can researchers optimize MAN2B2 antibody usage for dual labeling experiments?

Optimizing MAN2B2 antibody usage for dual labeling experiments requires careful consideration of multiple technical parameters to achieve specific, high-quality co-localization data. Primary antibody selection is critical, with researchers needing to choose MAN2B2 antibodies raised in different host species (e.g., rabbit anti-MAN2B2) from the antibody against the second target of interest to enable simultaneous detection without cross-reactivity . Sequential staining protocols may be preferable when both primary antibodies originate from the same species, involving complete detection of the first primary antibody before applying the second primary antibody, though this approach requires thorough blocking steps between detection cycles . Fluorophore selection for immunofluorescence applications should consider spectral separation to minimize bleed-through, with combinations such as FITC/Alexa Fluor 488 for MAN2B2 and Cy3/Alexa Fluor 594 for the co-marker providing good separation . Controls for dual labeling must include single-labeled samples to establish proper exposure settings and confirm absence of channel cross-talk, as well as secondary-only controls to verify absence of non-specific binding . For co-localization studies of MAN2B2 with lysosomal markers or glycosylation pathway components, proper fixation and permeabilization conditions are essential to preserve subcellular structures while allowing antibody access, with 4% paraformaldehyde fixation followed by 0.1-0.2% Triton X-100 permeabilization generally yielding good results . Quantitative co-localization analysis should employ appropriate statistical methods such as Pearson's correlation coefficient or Manders' overlap coefficient to objectively assess the degree of spatial correlation between MAN2B2 and other proteins of interest .

What computational approaches can complement MAN2B2 antibody-based research?

Computational approaches significantly enhance MAN2B2 antibody-based research by providing complementary analyses that expand interpretation and application of experimental data. Protein structure prediction algorithms can generate three-dimensional models of MAN2B2, enabling visualization of how genetic variants such as p.Asp38Asn or compound heterozygous variants (c.384G>T; c.926T>A) might affect protein folding, enzyme active sites, or epitope accessibility for antibody binding . Glycan structure prediction tools complement mass spectrometry data from MAN2B2-deficient samples, helping researchers interpret the accumulation of specific glycan structures like Man2GlcNac2 in patient fibroblasts and correlate these with MAN2B2 enzymatic function . Network analysis algorithms can integrate MAN2B2 protein interaction data obtained from co-immunoprecipitation experiments with known glycosylation pathway components, revealing potential functional relationships and compensatory mechanisms in disease states . Machine learning approaches applied to immunohistochemistry images can quantify MAN2B2 expression patterns across different tissues and disease states more objectively and comprehensively than manual scoring, potentially identifying subtle expression changes missed by visual inspection . For clinical research, multivariate statistical methods can correlate MAN2B2 expression data from antibody-based assays with patient clinical parameters, glycan profiles, and genetic information to identify biomarkers and stratify patient populations for personalized treatment approaches . Molecular dynamics simulations can model how MAN2B2 variants affect enzyme-substrate interactions at the atomic level, providing mechanistic insights into how specific mutations impact enzymatic function despite normal protein expression levels, as observed in some MAN2B2-CDG cases .