LMAN1 Antibody, Biotin conjugated

What is LMAN1 and what are its primary biological functions?

LMAN1 (Lectin Mannose-binding 1), also known as ERGIC-53, is a mannose-specific lectin that functions as a cargo receptor for ER-to-Golgi transport of selected proteins. It recognizes sugar residues of glycoproteins, glycolipids, or glycosylphosphatidyl inositol anchors and is involved in sorting or recycling of proteins and lipids . Recent research has identified LMAN1 as a receptor for house dust mite allergens , highlighting its role in allergic responses. Additionally, LMAN1 promotes trafficking of neuroreceptors including GABA ARs, 5-HT3 receptors, and nicotinic acetylcholine receptors , demonstrating its importance in the central nervous system.

What are the optimal conditions for using biotinylated LMAN1 antibodies in flow cytometry experiments?

For flow cytometry experiments using biotinylated LMAN1 antibodies, consider these methodological parameters:

• Cell preparation: Fix cells with 4% paraformaldehyde and permeabilize with 90% methanol for intracellular detection .

• Antibody dilution: For polyclonal biotinylated LMAN1 antibodies, a dilution range of 1:500-1:1000 is typically recommended for optimal signal-to-noise ratio .

• Secondary detection: Use streptavidin conjugated to a bright fluorophore (e.g., Alexa Fluor 488) at 1:2000 dilution .

• Controls: Include both isotype controls and cells without primary antibody incubation to establish background signal levels .

• Gating strategy: When analyzing LMAN1 expression in heterogeneous populations (like lung cells), use appropriate markers to distinguish between cell types, as LMAN1 expression varies significantly between populations (e.g., highly expressed on lung DCs ~95% vs. lower expression on alveolar macrophages) .

How should Western blot protocols be optimized when using biotinylated anti-LMAN1 antibodies?

When optimizing Western blot protocols for biotinylated anti-LMAN1 antibodies:

• Sample preparation: Use reducing conditions with 50μg of protein per lane .

• Gel parameters: Run on 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours .

• Transfer conditions: Transfer to nitrocellulose membrane at 150mA for 50-90 minutes .

• Blocking: Use 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Antibody incubation: When using biotinylated antibodies, they can be used directly without a secondary antibody step.

• Detection: Use streptavidin-HRP followed by enhanced chemiluminescent detection systems .

• Expected band size: LMAN1 should be detected at approximately 58kDa .

Include both positive controls (cells known to express LMAN1 such as HeLa or Jurkat) and negative controls (LMAN1 knockout cells if available) .

How can biotinylated LMAN1 antibodies be utilized in proximity-dependent labeling experiments?

Proximity-dependent labeling with biotinylated LMAN1 antibodies represents an advanced research application that can reveal protein-protein interactions and trafficking dynamics:

• BioID approach: Fusion of BirA* biotin ligase to SAR1B has been used to study LMAN1 interactions with COPII vesicles in cellular transport . This method revealed that approximately 50% of wild-type LMAN1 becomes biotinylated within 24 hours, providing quantitative insights into COPII recruitment dynamics.

• Pulse-chase experiments: Biotinylated LMAN1 antibodies can be used in conjunction with the RUSH (retention using selective hooks) system to visualize the concentrative sorting of LMAN1 by COPII . This technique allows for:

  • Synchronized release of LMAN1 from the ER

  • Real-time tracking of LMAN1 concentration in ER punctations

  • Visualization of LMAN1 disengagement from COPII and transport to the ERGIC

• Methodological considerations: When designing proximity labeling experiments, researchers should consider:

  • The location of lysine residues on LMAN1 for biotinylation (particularly in the 12-amino acid cytosolic tail)

  • Potential masking of these sites by interacting proteins

  • The impact of constitutively active SAR1B mutations on experimental outcomes

What is known about the glycan-independent interaction of LMAN1 with its cargo, and how can biotinylated antibodies help elucidate these mechanisms?

While LMAN1 typically functions as a mannose-specific lectin, recent research has revealed glycan-independent interactions with certain cargo proteins:

• Glycan-independent binding to GABA ARs: Co-immunoprecipitation experiments have shown that LMAN1 binds to GABA AR α1 subunits even when their N-glycosylation sites (Asn38 and Asn138) are mutated . Interestingly, the interaction with the non-glycosylated form was stronger than with the wild-type form.

• Domain mapping: Research has identified that:

  • N156A or D181A mutations in LMAN1, which disrupt mannose binding, do not affect interaction with GABA AR α1 subunits

  • The carbohydrate recognition domain (CRD) is still required, likely for structural integrity

  • Neither four β-sheets in the CRD nor the helix domain are required for α1 subunit binding

• Experimental approach using biotinylated antibodies: Biotinylated LMAN1 antibodies can be used to:

  • Immunoprecipitate LMAN1-cargo complexes for mass spectrometry analysis

  • Perform sequential immunoprecipitation to identify multiprotein complexes

  • Visualize co-localization of LMAN1 with non-glycosylated cargo in cellular compartments

This research has significant implications for understanding the versatility of LMAN1 in cargo recognition and transport.

How can non-specific binding be minimized when using biotinylated LMAN1 antibodies in immunohistochemistry?

Non-specific binding is a common challenge when using biotinylated antibodies in immunohistochemistry. To minimize this issue with LMAN1 detection:

• Antigen retrieval optimization: Heat-mediated antigen retrieval in citrate buffer (pH6) for 20 minutes has been validated for LMAN1 detection in paraffin-embedded tissue sections .

• Blocking protocol: Use 10% goat serum for blocking tissue sections before antibody incubation . This higher percentage of serum (compared to typical 5%) helps reduce background when using biotinylated antibodies.

• Endogenous biotin blocking: Use a commercial avidin/biotin blocking kit before applying biotinylated antibodies, as many tissues (especially liver and kidney) contain endogenous biotin that can lead to false-positive signals.

• Antibody concentration: For biotinylated polyclonal anti-LMAN1 antibodies, 1μg/ml has been validated for tissue section staining with minimal background .

• Secondary detection system: When using biotinylated primary antibodies, a streptavidin-based detection system should be used rather than a secondary antibody approach. Streptavidin-biotin-complex (SABC) with DAB as chromogen has been validated for LMAN1 detection .

How should researchers address conflicting data on LMAN1 expression patterns obtained using different detection methods?

When confronted with conflicting data on LMAN1 expression patterns:

• Validate antibody specificity: Confirm antibody specificity using LMAN1 knockout cells or tissues as negative controls . Western blot analysis showing a single band at 58kDa supports specificity.

• Compare detection methods: Different methods may reveal different aspects of LMAN1 biology:

  • Flow cytometry has revealed that LMAN1 is highly expressed on lung dendritic cell populations (~95%) and EpCAM+ airway epithelial cells (~80%)

  • Immunohistochemistry has demonstrated LMAN1 expression in diverse tissues including glioma, esophageal squamous cancer, placenta, and tonsil tissue

• Consider subcellular localization: LMAN1 cycles between the ER and Golgi, so its detection may vary depending on fixation methods and cell state:

  • In resting cells, LMAN1 shows classic ERGIC staining

  • During active transport, LMAN1 may show concentrated punctae on the ER

• Examine disease-related changes: In asthmatic individuals, peripheral dendritic cells show significant reduction in LMAN1 expression compared to healthy controls , which could explain some discrepancies in patient samples.

• Technical considerations: Create a comparison table documenting all methodological differences between experiments, including:

  • Fixation methods and duration

  • Antibody clones, dilutions, and incubation times

  • Detection systems and their sensitivities

  • Image acquisition parameters

How can biotinylated LMAN1 antibodies be used to investigate its role in allergic asthma pathogenesis?

To investigate LMAN1's role in allergic asthma using biotinylated antibodies:

• Ex vivo binding assays: Design experiments to measure binding of house dust mite (HDM) allergens to dendritic cells:

  • Flow cytometry-based cellular binding assays can be performed using biotinylated HDM extract or purified Der p 1 allergen

  • Cells with varying LMAN1 expression levels (overexpression, normal, underexpression) show corresponding levels of HDM binding

  • Secondary staining with streptavidin conjugates allows visualization of binding

• In vivo tracking: Study the dynamics of allergen uptake:

  • Administer fluorescent HDM intratracheally into mice

  • Use biotinylated LMAN1 antibodies to identify LMAN1-expressing cells

  • Flow cytometric analysis can identify specific lung cell populations (cDC2s, cDC1s, pDCs) that express LMAN1 and efficiently bind HDM

• Signaling pathway analysis: Investigate how LMAN1 regulates inflammatory responses:

  • LMAN1 overexpression downregulates NF-κB signaling in response to inflammatory cytokines or HDM

  • Dual-luciferase assay systems can be used to quantify this effect

  • Biotinylated antibodies can be used to immunoprecipitate LMAN1 complexes to identify interacting partners like FcRγ and SHP1

• Clinical correlations: Compare LMAN1 expression in patient samples:

  • Peripheral dendritic cells from asthmatic individuals show significantly reduced LMAN1 expression compared to healthy controls

  • Biotinylated antibodies can be used for more sensitive detection in limited clinical samples

What experimental approaches can determine if LMAN1-dependent cargo transport is affected by mutations in its glycan-binding domain?

To investigate how mutations in LMAN1's glycan-binding domain affect cargo transport:

• Cargo protein selection: Choose model cargo proteins with known dependency on LMAN1:

  • α1-antitrypsin (AAT) is a well-established LMAN1 cargo protein

  • Focus on the N-glycosylation site at N107, which is required for LMAN1-dependent AAT secretion

• LMAN1 mutant construction: Generate LMAN1 variants with targeted mutations:

  • N156A or D181A mutations disrupt mannose binding

  • Study the CRD domain and its role in cargo binding

  • The FF motif in LMAN1's C-terminus is required for COPII interaction

• Cellular assay design: Use complementary approaches to assess transport:

  • CHX chase experiments can track secretion rates in WT versus LMAN1 knockout cells

  • Surface biotinylation can quantify plasma membrane delivery of cargo

  • Co-immunoprecipitation with biotinylated antibodies can assess physical interactions between LMAN1 mutants and cargo proteins

• In vitro binding assays: Directly measure binding of purified components:

  • Express and purify LMAN1 using D-mannose agarose beads

  • Test binding to cargo proteins with and without glycosylation

  • Compare wild-type and mutant LMAN1 binding affinities

• Data analysis approach:

This comprehensive approach can distinguish between glycan-dependent and glycan-independent mechanisms of LMAN1-mediated cargo transport.

How can biotinylated LMAN1 antibodies contribute to therapeutic development for allergic diseases?

Biotinylated LMAN1 antibodies can facilitate therapeutic development for allergic diseases through several research approaches:

• Target validation: LMAN1 has been identified as a receptor for house dust mite allergens , making it a potential therapeutic target. Biotinylated antibodies can:

  • Confirm LMAN1 expression in relevant patient tissues

  • Quantify changes in LMAN1 levels during disease progression

  • Assess correlation between LMAN1 expression and disease severity

• Mechanism exploration: Understanding how LMAN1 regulates allergic responses:

  • LMAN1 overexpression downregulates NF-κB signaling in response to inflammatory stimuli

  • HDM promotes binding of LMAN1 to FcRγ and recruitment of SHP1

  • Biotinylated antibodies can help isolate and characterize these protein complexes

• Drug screening platforms: Development of high-throughput screening systems:

  • Cell-based assays using biotinylated LMAN1 antibodies to detect changes in expression or localization

  • Competition assays to identify compounds that disrupt LMAN1-allergen interactions

  • Conformational antibodies that distinguish between different functional states of LMAN1

• Biomarker development: The reduction of LMAN1 in peripheral DCs of asthmatic individuals suggests potential as a biomarker:

  • Biotinylated antibodies can be used in multiplexed biomarker panels

  • Development of point-of-care diagnostics using sensitive detection methods

  • Longitudinal studies to determine if LMAN1 levels predict treatment response

What are the considerations for using biotinylated LMAN1 antibodies in multiplex imaging of trafficking pathways?

When designing multiplex imaging experiments to study LMAN1 trafficking:

• Conjugation chemistry: Consider the biotinylation method carefully:

  • N-succinimidyl ester crosslinking to ε-amino groups of lysine residues is commonly used

  • Site-specific biotinylation methods may be preferable to maintain antibody function

  • The degree of biotinylation should be optimized (typically 3-5 biotin molecules per antibody)

• Multicolor strategy: For multiplexed imaging:

  • Use streptavidin conjugated to spectrally distinct fluorophores

  • Consider sequential detection using streptavidin conjugates with different fluorophores

  • Implement tyramide signal amplification for low-abundance targets

• Temporal dynamics: Capturing LMAN1's cycling between the ER and Golgi:

  • Design pulse-chase experiments using the RUSH system

  • Track LMAN1 concentration in ER punctations and subsequent transport to ERGIC

  • Compare wild-type LMAN1 with mutants (e.g., FF/AA motif that eliminates COPII interaction)

• Co-visualization with multiple markers: Important markers to include:

  • SEC24A to identify COPII-coated vesicles

  • MCFD2 to visualize the LMAN1-MCFD2 complex formation

  • Cargo proteins such as coagulation factors V and VIII, AAT, or neuroreceptors

• Advanced imaging techniques:

  • Super-resolution microscopy to resolve vesicular structures below the diffraction limit

  • Light sheet microscopy for 3D visualization of trafficking in thick specimens

  • Live-cell imaging to capture dynamic interactions between LMAN1 and its cargo