LMAN1 Antibody, HRP conjugated

What is LMAN1 and what are its key functions in cellular processes?

LMAN1 (Lectin Mannose-binding 1) is a mannose-specific lectin that plays several important roles in cellular transport and immune function. It recognizes sugar residues of glycoproteins, glycolipids, and glycosylphosphatidyl inositol anchors, and is involved in the sorting or recycling of proteins and lipids. The LMAN1-MCFD2 complex forms a specific cargo receptor for ER-to-Golgi transport of selected proteins . Recent research has revealed that LMAN1 also functions as a receptor for house dust mite (HDM) allergens, binding directly to allergen extracts and purified major HDM allergens such as Der p 1 . LMAN1 is expressed on the surface of dendritic cells and airway epithelial cells, suggesting its role in immune recognition and allergen sensing.

What is the structural composition of LMAN1 protein?

LMAN1 has a complex structure consisting of several distinct domains, each with specific functions:

• N-terminal signal sequence

• Carbohydrate recognition domain (CRD)

• Helical or stalk domain

• Transmembrane region

• Short cytoplasmic region (12 amino acids)

The carbohydrate recognition domain (CRD) is particularly important as it is responsible for binding to house dust mite allergens, with the N156 residue within this domain being critical for calcium-dependent binding to mannosylated cargo . LMAN1 exists endogenously as homohexamers, which affects its binding properties and function .

How does an HRP-conjugated LMAN1 antibody differ from unconjugated versions?

HRP (Horseradish Peroxidase) conjugated LMAN1 antibodies offer direct detection capabilities without requiring secondary antibodies. The HRP enzyme catalyzes a reaction that produces a detectable signal when exposed to an appropriate substrate. This direct detection system can reduce background noise, decrease experimental time, and potentially increase sensitivity compared to unconjugated antibodies that require secondary antibody detection . The conjugation process maintains the LMAN1 antibody's specificity while adding functionality through the covalently attached HRP enzyme. When selecting between conjugated and unconjugated versions, researchers must consider factors such as signal amplification requirements, detection method compatibility, and potential steric hindrance effects of the HRP moiety on antigen binding.

What are the optimal conditions for western blot detection using LMAN1 antibody?

For optimal western blot detection using LMAN1 antibody, the following protocol is recommended:

• Antibody concentration: Use LMAN1 antibody at 1.25 μg/mL for detection .

• Secondary antibody dilution: If using an unconjugated primary LMAN1 antibody, the HRP-conjugated secondary antibody should be diluted 1:50,000-100,000 .

• Sample preparation: Lyse cells completely in RIPA buffer with protease inhibitors and heat samples at 95°C for 5 minutes in reducing buffer.

• Electrophoresis conditions: Run samples on 10-12% SDS-PAGE gel at 100-120V.

• Transfer parameters: Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes.

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Antibody incubation: Incubate with primary antibody overnight at 4°C, followed by multiple TBST washes.

• Detection: For HRP-conjugated LMAN1 antibody, proceed directly to enhanced chemiluminescence (ECL) detection after washes.

The expected band for LMAN1 should appear at approximately 53 kDa, which corresponds to the ER-Golgi intermediate compartment 53 kDa protein .

How can LMAN1 antibodies be utilized in immunoprecipitation studies of protein-protein interactions?

LMAN1 antibodies can be effectively utilized in immunoprecipitation (IP) studies to investigate protein-protein interactions, particularly focusing on LMAN1's role in forming complexes with other proteins. Research has demonstrated successful applications in studying interactions between LMAN1 and adaptor proteins:

• Co-immunoprecipitation protocol:

  • Lyse cells in non-denaturing lysis buffer (1% NP-40, 150mM NaCl, 50mM Tris pH 8.0, protease inhibitors)

  • Pre-clear lysate with protein A/G beads

  • Incubate with LMAN1 antibody (2-5μg) overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours

  • Wash extensively with lysis buffer

  • Elute and analyze by western blot

• Detection of protein interactions: This method has successfully revealed interactions between LMAN1 and FcRγ, confirming that HDM stimulation promotes binding of LMAN1 and FcRγ at around 60 minutes in control cells .

• Signaling complex identification: IP studies have shown that LMAN1-FcRγ interactions coincide with the recruitment of SHP1, a known negative regulator of FcRγ and C-type lectin receptor (CLR)-mediated pathways .

The specificity of these interactions can be confirmed by performing reciprocal immunoprecipitation experiments, where immunoprecipitation of LMAN1 via its tag pulls down the interacting protein, and vice versa .

What considerations are important when designing flow cytometry experiments with LMAN1 antibodies?

When designing flow cytometry experiments with LMAN1 antibodies, several critical factors must be considered to ensure valid and reproducible results:

• Surface versus intracellular staining protocols:

  • For surface LMAN1: Use non-permeabilizing conditions and shorter incubation times

  • For total LMAN1: Include permeabilization step to detect intracellular pools

• Antibody validation:

  • Confirm specificity using positive controls (cells known to express LMAN1) and negative controls (LMAN1 knockdown cells)

  • Verify detection using both HRP-conjugated and unconjugated formats with appropriate secondary antibodies

• Multi-parameter analysis considerations:

  • LMAN1 is highly expressed on lung dendritic cell populations (~95% of cDC2s, cDC1s, and pDCs)

  • Also expressed on ~80% of EpCAM+ airway epithelial cells

  • Lower expression on alveolar macrophages

• Gating strategy for specific cell populations:

  • When analyzing LMAN1 expression in relation to HDM binding, consider both HDM^lo and HDM^hi populations

  • LMAN1 expression correlates positively with efficient HDM binding/uptake

• Sample preparation optimizations:

  • Fresh vs. fixed cells (fixation may affect epitope recognition)

  • Buffer composition (calcium concentration affects LMAN1 binding properties)

  • Antibody concentration titration to determine optimal signal-to-noise ratio

During analysis, researchers should note that LMAN1 expression varies significantly between cell types and disease states, with peripheral dendritic cells from asthmatic individuals showing reduced LMAN1 expression compared to healthy controls .

How does LMAN1 regulate NF-κB signaling in response to allergens, and how can this be experimentally measured?

LMAN1 functions as a negative regulator of NF-κB signaling in response to allergens, particularly house dust mite (HDM) allergens. This regulatory relationship can be experimentally assessed through several methodological approaches:

• Dual-luciferase reporter assay:

  • Transfect cells with NF-κB reporter construct, control Renilla luciferase, and varying amounts of LMAN1 expression vector

  • Stimulate with HDM or TNF-α

  • Measure luciferase activity

  • Results show dose-dependent reduction in NF-κB activation with increasing LMAN1 expression

• Western blot analysis of signaling components:

  • Compare phosphorylation status of IκBα in LMAN1-overexpressing versus control cells after HDM stimulation

  • LMAN1 overexpression reduces phosphorylation of IκBα

  • LMAN1 knockdown enhances phosphorylation of IκBα

• Mechanistic pathway analysis:

  • HDM stimulation promotes binding of LMAN1 to FcRγ

  • This interaction coincides with recruitment of SHP1, a negative regulator of signaling

  • The timing of these interactions correlates with downregulation of NF-κB activation

• Experimental controls:

  • Compare HDM versus LPS stimulation (LMAN1 regulatory effects are specific to HDM)

  • Different kinetics of NF-κB activation are observed with different stimuli

  • LMAN1 overexpression does not affect LPS-induced NF-κB activation

These findings suggest that LMAN1 functions as part of a regulatory feedback mechanism, potentially limiting inflammatory responses to allergens through recruitment of negative regulators like SHP1 to signaling complexes.

What cellular distribution patterns of LMAN1 can be detected using immunofluorescence techniques?

LMAN1 exhibits distinct cellular distribution patterns that can be visualized using immunofluorescence techniques. Proper immunofluorescence protocols reveal important insights into LMAN1's localization and functional domains:

• Subcellular localization patterns:

  • Primary localization: ER-Golgi intermediate compartment (ERGIC) and early Golgi

  • Secondary localization: Cell surface (particularly in dendritic cells)

  • Confocal microscopy on unpermeabilized dendritic cells has shown colocalization between LMAN1 and HDM allergen Der p 1 at discrete regions on the cell surface

• Protocol optimization for different localizations:

  • For ER/Golgi localization: Permeabilization with 0.1% Triton X-100

  • For surface localization: No permeabilization, staining of live cells at 4°C

  • Fixation: 4% paraformaldehyde (PFA) for 15 minutes

• Co-localization studies:

  • LMAN1 co-localizes with:

    • ER markers (calnexin, PDI) in the ER

    • ERGIC-53 in the ER-Golgi intermediate compartment

    • GM130 in the cis-Golgi

    • Der p 1 at the cell surface

• Cell-type specific distribution:

  • Dendritic cells: Both intracellular and surface expression

  • Airway epithelial cells: Predominantly surface expression (~80% of EpCAM+ cells)

  • T cells: Present on ~15% of recruited cells in allergen challenge models

  • B cells and neutrophils: Present on ~37% and ~42% respectively in BAL fluid after allergen challenge

These localization patterns provide insight into LMAN1's dual role as both an intracellular cargo receptor and a cell surface receptor for allergens, with potential implications for targeting allergic disease pathways.

How does domain structure affect LMAN1 function, and what experimental approaches can assess domain-specific activity?

The domain structure of LMAN1 critically influences its various functions, with specific domains mediating different aspects of its activity. Several experimental approaches can elucidate domain-specific contributions:

• Domain-deletion mutant analysis:

  • LMAN1 lacking the carbohydrate recognition domain (ΔCRD)

  • LMAN1 lacking the helical domain (ΔHelix)

  • Point mutation of the N156 residue to alanine (N156A) within the CRD domain

  • Results show that the CRD domain is essential for binding to HDM allergens

• Domain-specific binding assays:

  • Cell-based binding assays using LMAN1 constructs with various domain mutations

  • Purified domain protein interactions with ligands (HDM allergens, cargo proteins)

  • Surface plasmon resonance (SPR) to measure binding kinetics and affinity

• Functional rescue experiments:

  • Expression of specific domains in LMAN1-deficient cells

  • Assessment of which domains can restore:

    • Cargo transport between ER and Golgi

    • HDM allergen binding

    • NF-κB signaling regulation

• Structure-function relationship analysis:

  • The carbohydrate recognition domain (CRD) mediates mannose-dependent binding

  • The N156 residue within the CRD is critical for calcium-dependent recognition

  • The helical domain contributes to oligomerization of LMAN1 into homohexamers

  • The short cytoplasmic domain (12 amino acids) lacks obvious signaling motifs but may interact with adaptor proteins like FcRγ

Understanding these domain-specific functions helps explain how LMAN1 can simultaneously act as a cargo receptor for ER-to-Golgi transport and as a cell surface receptor for allergens, with important implications for both normal cellular function and pathological conditions like allergic asthma.

What factors can affect LMAN1 antibody specificity, and how can cross-reactivity be assessed?

Several factors can influence LMAN1 antibody specificity, and systematic assessment of cross-reactivity is essential for generating reliable research data:

• Epitope location considerations:

  • Antibodies targeting different domains (CRD vs. helical vs. cytoplasmic) may exhibit different specificity profiles

  • Conformational changes in LMAN1 (calcium-dependent) can affect epitope accessibility

  • HRP conjugation may sterically hinder certain epitopes

• Cross-reactivity assessment methods:

  • Western blot analysis using tissues from multiple species (human, mouse, rat, dog)

  • Immunoprecipitation followed by mass spectrometry to identify all pulled-down proteins

  • Comparative staining in LMAN1 knockout/knockdown versus wildtype cells

  • Pre-absorption with recombinant LMAN1 protein to confirm specificity

• Homology considerations:

  • LMAN1 shares sequence similarity with other lectins and mannose-binding proteins

  • Testing against related family members is crucial

  • Specific validation in each experimental system is recommended

• Application-specific validation:

  Application	Validation Approach	Expected Results
  Western Blot	Band size verification	Single band at ~53 kDa
  Flow Cytometry	Comparison to isotype control	Clear separation of positive/negative populations
  Immunohistochemistry	Peptide competition	Elimination of specific staining
  ELISA	Titration curve with recombinant protein	Linear detection range at 1:1562500 dilution

• Species reactivity verification:

  • The antibody has been verified to react with human, mouse, rat, and dog LMAN1

  • Cross-reactivity prediction based on sequence homology should be experimentally confirmed

Robust validation across multiple applications ensures that experimental observations genuinely reflect LMAN1 biology rather than artifacts of cross-reactivity.

How can researchers optimize LMAN1 antibody reconstitution and storage to maintain activity?

Proper reconstitution and storage of LMAN1 antibodies are critical for maintaining optimal activity and ensuring reproducible experimental results. Following these methodological guidelines can significantly improve antibody performance:

• Reconstitution protocol:

  • For lyophilized antibody: Add 100 μL of distilled water to achieve a final concentration of 1 mg/mL

  • Allow complete dissolution at room temperature (15-20 minutes)

  • Avoid vigorous shaking or vortexing which can cause protein denaturation

  • The reconstitution buffer typically contains PBS with 2% sucrose as a stabilizer

• Storage recommendations:

  • Aliquot into small volumes (10-20 μL) to avoid repeated freeze-thaw cycles

  • Store at -20°C or below for long-term stability

  • For working solutions, store at 4°C for up to 2 weeks

  • Avoid more than 3 freeze-thaw cycles which can reduce activity by up to 30% per cycle

• Stability enhancement strategies:

  Additive	Concentration	Benefit
  BSA	1-5 mg/mL	Prevents adsorption to tubes
  Glycerol	30-50%	Prevents freezing at -20°C
  Sodium azide	0.02%	Prevents microbial growth
  Trehalose	5%	Stabilizes protein structure

• Activity monitoring approaches:

  • Perform regular activity tests using positive control samples

  • Compare signal intensity to previous lots or freshly reconstituted antibody

  • For HRP-conjugated antibodies, assess enzyme activity using standard TMB substrate

• Troubleshooting reduced activity:

  • If activity decreases, verify pH stability (optimal range: 6.5-7.5)

  • Check for precipitates or turbidity which indicate protein aggregation

  • Consider using carrier proteins or stabilizers if repeated activity loss occurs

  • HRP conjugates are particularly sensitive to oxidizing agents and extreme pH

Following these guidelines ensures maximum retention of antibody activity and extends the useful life of valuable research reagents.

What controls should be included when using LMAN1 antibodies in advanced experimental designs?

Implementing appropriate controls is essential when using LMAN1 antibodies to ensure data validity and reproducibility, particularly in complex experimental designs:

• Primary antibody controls:

  • Isotype control: Matched concentration of non-specific immunoglobulin from same species

  • Absorbed control: Primary antibody pre-incubated with excess recombinant LMAN1

  • Concentration gradient: Titration to determine optimal antibody concentration

• Genetic manipulation controls:

  • LMAN1 knockdown/knockout cells: Demonstrate antibody specificity

  • LMAN1 overexpression: Verify signal intensity correlation with expression level

  • Domain mutants: Confirm epitope specificity (particularly important for CRD domain)

• Application-specific controls:

  Application	Positive Control	Negative Control	Technical Control
  Western Blot	Lung or dendritic cell lysate	LMAN1 knockout lysate	Loading control (β-actin)
  Flow Cytometry	Lung DCs (~95% positive)	LMAN1 knockdown cells	Fluorescence minus one (FMO)
  Immunoprecipitation	Input sample (pre-IP)	IP with non-specific IgG	Heavy chain detection control
  Functional Assays	HDM stimulation	TNF-α or LPS stimulation	Pathway inhibitor controls

• Signal validation controls:

  • For HRP-conjugated antibodies: Substrate-only control to assess background

  • Hydrogen peroxide pre-treatment: Inactivates endogenous peroxidases

  • Secondary-only control: Detects non-specific binding of detection system

• Physiological relevance controls:

  • Multi-species comparison: Verify conservation of expression patterns

  • Cell activation status: Compare resting vs. stimulated cells (HDM challenge)

  • Pathological condition comparison: Healthy vs. asthmatic samples

These comprehensive controls ensure that experimental observations reflect authentic LMAN1 biology rather than technical artifacts or non-specific interactions, particularly important when investigating LMAN1's dual roles in cargo transport and allergen recognition.

How does LMAN1 expression change in allergic conditions, and what methodologies can assess these alterations?

LMAN1 expression undergoes significant changes in allergic conditions, particularly in asthma, with important implications for disease pathophysiology. Several methodological approaches can effectively assess these alterations:

• Flow cytometric quantification:

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

  • Cell-specific analysis reveals differential expression patterns:

    • ~95% of lung dendritic cells express LMAN1 in healthy conditions

    • ~80% of airway epithelial cells express LMAN1 basally

• Transcriptomic analysis approaches:

  • RNA-seq of sorted cell populations from control vs. allergen-challenged lungs

  • Single-cell RNA sequencing to identify cell-specific expression changes

  • qRT-PCR validation of LMAN1 transcript levels in clinical samples

• Protein quantification methods:

  Method	Sample Type	Measurement Parameter
  Western Blot	Tissue/cell lysates	Total LMAN1 protein levels
  Flow Cytometry	Single cell suspensions	Cell-specific expression
  Mass Spectrometry	Bronchoalveolar lavage	LMAN1 protein abundance
  ELISA	Serum/plasma	Soluble LMAN1 fragments

• Animal model validation:

  • House dust mite (HDM) asthma models show altered LMAN1 expression

  • Assessment of recruited inflammatory cells:

    • T cells: ~15% express LMAN1

    • B cells: ~37% express LMAN1

    • Neutrophils: ~42% express LMAN1

• Clinical correlation approaches:

  • Stratification of patients by asthma severity

  • Correlation with allergen-specific IgE levels

  • Longitudinal monitoring during allergen challenge or immunotherapy

These methodologies provide complementary insights into how LMAN1 expression changes during allergic responses, with potential implications for biomarker development and therapeutic targeting. The consistent finding of reduced LMAN1 expression in asthmatic conditions suggests its role as a negative regulator of allergic inflammation.

What is the relationship between LMAN1 and house dust mite allergen recognition, and how can this interaction be experimentally manipulated?

The relationship between LMAN1 and house dust mite (HDM) allergen recognition represents a significant advance in understanding allergen sensing mechanisms. This interaction can be experimentally manipulated through several methodological approaches:

• Direct binding assays:

  • Affinity-purified FLAG-tagged LMAN1 shows dose-dependent binding to both total HDM extract (D. pteronyssinus) and purified Der p 1 allergen

  • Similar binding patterns observed with D. farinae extracts, indicating cross-species recognition

• Cellular binding experiments:

  • Flow cytometry-based assays using biotinylated HDM extract or purified allergens

  • LMAN1 overexpression increases HDM binding while knockdown reduces binding

  • Confocal microscopy demonstrates colocalization between LMAN1 and Der p 1 at discrete regions on the cell surface

• Structure-based manipulation approaches:

  Manipulation	Method	Observed Effect
  Domain deletion	ΔCRD construct	Abolishes HDM binding
  Point mutation	N156A mutation	Disrupts calcium-dependent binding
  Antibody blocking	Anti-LMAN1 antibodies	Enhances NF-κB signaling
  siRNA knockdown	Electroporation	Increases NF-κB activation

• In vivo interaction assessment:

  • Intratracheal administration of fluorescent HDM reveals HDM^hi and HDM^lo cell populations

  • LMAN1 expression correlates with efficient HDM binding/uptake

  • Multiple cell types interact with HDM via LMAN1: dendritic cells, airway epithelial cells, macrophages

• Signaling pathway manipulation:

  • HDM stimulation promotes binding of LMAN1 to FcRγ at approximately 60 minutes

  • This interaction coincides with SHP1 recruitment, a negative regulator of signaling

  • Blocking this interaction enhances inflammatory responses

These experimental approaches reveal LMAN1 as a critical receptor for allergen recognition with immunomodulatory functions. The ability to manipulate this interaction opens possibilities for therapeutic interventions targeting allergic diseases at the recognition level rather than downstream inflammatory pathways.

How can researchers investigate potential therapeutic applications targeting LMAN1 in allergic diseases?

Investigating LMAN1's therapeutic potential in allergic diseases requires strategic research approaches that build on its newly discovered role as an allergen receptor and immune modulator:

• LMAN1 modulation strategies:

  • Recombinant LMAN1-Fc fusion proteins to competitively inhibit allergen binding

  • Small molecule enhancers of LMAN1 expression or stability

  • Peptide mimetics targeting the LMAN1-FcRγ-SHP1 signaling complex

  • Gene therapy approaches to restore LMAN1 levels in dendritic cells

• Preclinical model testing framework:

  • HDM-induced asthma models in wild-type vs. LMAN1-deficient mice

  • Therapeutic intervention timepoints:

    • Preventive (before allergen exposure)

    • Early intervention (during sensitization)

    • Therapeutic (established disease)

  • Readouts: airway hyperresponsiveness, inflammation, IgE levels, cytokine production

• Biomarker development pathway:

  Biomarker Type	Sample Source	Clinical Correlation
  LMAN1 expression	Peripheral DCs	Disease severity
  Soluble LMAN1	Bronchoalveolar lavage	Local inflammation
  LMAN1 polymorphisms	Genetic screening	Disease susceptibility
  LMAN1-allergen complexes	Serum	Allergen exposure

• Translational research approaches:

  • Ex vivo testing using patient-derived cells

  • Comparison of LMAN1 expression before and after allergen immunotherapy

  • Correlation of LMAN1 expression with treatment response

  • Development of personalized medicine approaches based on LMAN1 status

• Drug development considerations:

  • Target validation using conditional knockout models

  • Screening assays based on LMAN1-HDM binding inhibition

  • Safety assessment considering LMAN1's role in cargo transport

  • Delivery systems specifically targeting dendritic cells or airway epithelium

These research pathways could lead to novel therapeutic strategies targeting the initial allergen recognition events rather than downstream inflammatory cascades. The reduced expression of LMAN1 in asthmatic patients suggests that restoring its levels or function might help reestablish immune homeostasis in allergic conditions . Such approaches would represent a paradigm shift from symptom management to addressing fundamental mechanisms of allergic disease.