LMAN1 Antibody, FITC conjugated

What is LMAN1 and what are its key cellular functions?

LMAN1 (also known as ERGIC-53) is a type 1-transmembrane protein that forms a complex with MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). It functions as a mannose-specific lectin that recognizes sugar residues of glycoproteins, glycolipids, or glycosylphosphatidyl inositol anchors. The LMAN1-MCFD2 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins, including coagulation factors V and VIII . Recent research has identified LMAN1 as a receptor for house dust mite (HDM) allergens, where it plays a regulatory role in allergic responses by downregulating NF-κB signaling in response to inflammatory cytokines or HDM exposure .

What are the technical specifications of LMAN1 Antibody, FITC conjugated?

LMAN1 Antibody, FITC conjugated (catalog number CSB-PA012991LC01HU) is a rabbit polyclonal antibody specifically reactive to human LMAN1 . The key specifications are summarized in the following table:

How is LMAN1 expressed across different tissues and cell types?

LMAN1 shows differential expression across tissues, with the highest levels observed in professional secretory organs like pancreas and salivary gland, while tissues with lower secretion activities such as heart, muscle, and brain display reduced expression . In lung tissue, LMAN1 is highly expressed on various dendritic cell populations (cDC2s, cDC1s, and pDCs) at approximately 95% and on airway epithelial cells (AECs) at around 80% . During inflammatory conditions like asthma, LMAN1 can also be detected on recruited immune cells, including a smaller population of T cells (~15%), B cells (~37%), and neutrophils (~42%) . This expression pattern has significant implications for both protein trafficking studies and investigations into allergic airway diseases.

What controls should be included when using LMAN1 Antibody, FITC conjugated in flow cytometry experiments?

When designing flow cytometry experiments with LMAN1 Antibody, FITC conjugated, implement the following control strategy:

• Antibody controls:

  • Isotype control: FITC-conjugated rabbit IgG polyclonal antibody at equal concentration

  • Unstained cells to establish autofluorescence baseline

  • Fluorescence Minus One (FMO) controls if using multiple antibodies

• Biological controls:

  • Positive control: Cell types known to express high levels of LMAN1 (e.g., dendritic cells, as ~95% of lung DCs express LMAN1)

  • Negative control: LMAN1 knockdown cells or tissues with minimal expression

  • Comparative analysis of HDM-high and HDM-low binding populations, as HDM-high populations correlate with higher LMAN1 expression

• Procedural controls:

  • Antibody titration to determine optimal working concentration

  • Dead cell exclusion dye to prevent false positives

  • Compensation controls if using multiple fluorophores

How can researchers optimize detection of both surface and intracellular LMAN1?

LMAN1 has dual localization, functioning both as an intracellular cargo receptor and as a surface receptor for allergens . To optimize detection of both populations:

For surface LMAN1:

• Use fresh, unfixed cells when possible

• Maintain samples at 4°C throughout staining to prevent internalization

• Include sodium azide (0.1%) in staining buffer to inhibit metabolic processes

• Use gentle cell dissociation methods (e.g., EDTA-based rather than trypsin) to preserve surface epitopes

• Validate surface staining with non-permeabilized flow cytometry controls

For intracellular LMAN1:

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes

• Permeabilize with 0.1% saponin or 0.1% Triton X-100

• Extend primary antibody incubation time (1-2 hours or overnight at 4°C)

• Include saponin in all wash buffers to maintain permeabilization

• Consider signal amplification methods for detecting lower expression levels

What approaches can resolve contradictory data regarding LMAN1 expression levels?

When confronted with conflicting data regarding LMAN1 expression, implement these methodological approaches:

• Multi-platform validation:

  • Confirm expression using complementary techniques (RT-qPCR, Western blot, immunofluorescence)

  • Compare results from multiple LMAN1 antibodies targeting different epitopes

  • Include genetic validation (siRNA knockdown) to verify specificity

• Context-dependent evaluation:

  • Control for activation state, as LMAN1 expression may change upon cell stimulation or allergen exposure

  • Assess experimental timing, as LMAN1 may show temporal dynamics after stimulation

  • Compare healthy versus diseased samples, as LMAN1 is reduced in asthmatic individuals' dendritic cells

• Technical optimization:

  • Standardize fixation and permeabilization protocols

  • Control for sample handling (avoid repeated freeze-thaw of antibody)

  • Implement quantitative flow cytometry with calibration beads to obtain absolute expression values

How can researchers investigate LMAN1's role in coagulation factor transport?

To study LMAN1's role in coagulation factor transport, researchers can implement these methodological approaches:

• Secretion assays:

  • Measure Factor V and Factor VIII activity in culture supernatants of control versus LMAN1-depleted cells

  • Compare with mouse models, as LMAN1-deficient mice show ~50% reduction in plasma FV and FVIII levels

  • Use pulse-chase experiments to track the kinetics of factor secretion

• Co-localization studies:

  • Perform dual immunostaining of LMAN1 and coagulation factors in the secretory pathway

  • Use super-resolution microscopy to visualize transport intermediates

  • Implement live-cell imaging with tagged constructs to track trafficking in real-time

• Interaction analyses:

  • Conduct co-immunoprecipitation experiments to confirm LMAN1-factor interactions

  • Investigate dependence on MCFD2, as this cofactor is required for coagulation factor binding

  • Map interaction domains through deletion mutants or peptide competition assays

What experimental approaches can track LMAN1 cycling between ER and ERGIC?

To investigate LMAN1 trafficking dynamics between cellular compartments:

• Live-cell imaging techniques:

  • Transfect cells with LMAN1-GFP fusion constructs for real-time visualization

  • Implement photoactivatable or photoconvertible tags for pulse-chase imaging

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility rates

• Subcellular fractionation approaches:

  • Isolate ER, ERGIC, and Golgi fractions using differential centrifugation

  • Quantify LMAN1 distribution across fractions via Western blotting

  • Compare wild-type LMAN1 versus mutants lacking ER retrieval motifs (KKFF)

• Cargo-based trafficking assays:

  • Monitor co-trafficking of LMAN1 with known cargo molecules

  • Implement temperature blocks (15°C) to accumulate proteins in the ERGIC

  • Use brefeldin A to disrupt anterograde transport and assess LMAN1 redistribution

How can LMAN1 Antibody be utilized to study its newly identified role as a house dust mite receptor?

The discovery of LMAN1 as a house dust mite (HDM) allergen receptor opens new research avenues . Methodological approaches include:

• Binding characterization:

  • Use flow cytometry with FITC-conjugated LMAN1 antibody to identify and quantify LMAN1-expressing cells in airway samples

  • Perform competitive binding assays between unlabeled antibody and fluorescently-labeled HDM allergens

  • Conduct dose-response binding experiments with purified HDM allergens on LMAN1-expressing versus knockdown cells

• Functional studies:

  • Pre-block LMAN1 with antibodies before HDM challenge to assess impact on NF-κB activation and inflammatory responses

  • Compare phosphorylation of IκBα in LMAN1-overexpressing versus control cells after HDM stimulation, as LMAN1 overexpression reduces IκBα phosphorylation

  • Analyze cytokine/chemokine production in cells with different LMAN1 expression levels following HDM exposure

• In vivo experimental design:

  • Compare HDM binding capacity of lung cells in wild-type versus LMAN1-deficient mice

  • Assess the correlation between LMAN1 expression and HDM binding efficiency across different cell populations, as HDM-high cells show higher LMAN1 expression

  • Examine DC activation and migration to draining lymph nodes in the presence of LMAN1-blocking antibodies

What methodological approaches can investigate LMAN1's role in regulating NF-κB signaling?

To explore LMAN1's newly identified role in downregulating inflammatory signaling:

• Signaling pathway analysis:

  • Compare NF-κB activation using dual-luciferase reporter assays in control versus LMAN1-overexpressing cells, as increased LMAN1 expression shows dose-dependent reduction in NF-κB activation

  • Monitor phosphorylation kinetics of IκBα using phospho-specific antibodies and Western blotting

  • Assess nuclear translocation of NF-κB subunits via cellular fractionation or imaging approaches

• Adaptor protein recruitment studies:

  • Investigate LMAN1 interaction with FcRγ through reciprocal co-immunoprecipitation experiments

  • Analyze recruitment of SHP1 phosphatase following HDM stimulation

  • Map critical domains required for these interactions using deletion mutants

• Experimental validation:

  • Compare effects in multiple cell types including dendritic cells and epithelial cells

  • Implement LMAN1 blocking antibodies to assess enhancement of phospho-IκBα and phospho-Syk

  • Utilize both inflammatory cytokine (TNF-α) and allergen (HDM) stimulation to confirm consistency of findings

How can researchers investigate alterations in LMAN1 expression in asthmatic patients?

To study LMAN1 dysregulation in asthma:

• Clinical sample analysis:

  • Compare LMAN1 expression on peripheral blood DCs from asthmatic versus healthy subjects, as asthmatic individuals show reduced LMAN1 expression

  • Perform flow cytometric quantification of LMAN1 on multiple immune cell subsets

  • Correlate LMAN1 expression levels with clinical measures of disease severity

• Functional assessment:

  • Compare HDM binding capacity of cells from asthmatic versus healthy donors

  • Analyze NF-κB activation in response to HDM in cells with different LMAN1 expression levels

  • Assess inflammatory mediator production in ex vivo stimulated cells

• Genetic and epigenetic investigations:

  • Sequence LMAN1 locus to identify potential polymorphisms associated with asthma

  • Analyze promoter methylation status to assess epigenetic regulation

  • Perform transcription factor binding studies to identify regulatory mechanisms

What are common challenges when using LMAN1 Antibody, FITC conjugated, and how can they be addressed?

Researchers frequently encounter these challenges when working with LMAN1 Antibody, FITC conjugated:

• Signal fading:

  • Store antibody protected from light at recommended temperatures (-20°C or -80°C)

  • Add anti-fade reagents to mounting media for microscopy applications

  • Minimize exposure to excitation light during imaging

  • Avoid repeated freeze-thaw cycles which can degrade the FITC conjugate

• High background:

  • Optimize blocking conditions (5% normal serum for 1-2 hours)

  • Include 0.1% Tween-20 in wash buffers

  • Perform additional washing steps

  • Filter antibody solution through a 0.22μm filter before use to remove aggregates

• Weak signal:

  • Test multiple fixation protocols as some may mask the epitope

  • Implement signal amplification methods

  • Increase antibody concentration within recommended range

  • Extend incubation time (overnight at 4°C)

How can LMAN1 Antibody, FITC conjugated be integrated into multicolor flow cytometry panels?

When designing multicolor panels incorporating LMAN1 Antibody, FITC conjugated:

• Panel design considerations:

  • Place FITC (excited by 488nm laser) in a channel for moderately expressed markers

  • Avoid fluorophores with spectral overlap with FITC (e.g., PE) for critical markers

  • Reserve brighter fluorophores (APC, PE-Cy7) for low-expression markers

• Optimization strategy:

  • Perform antibody titration to determine optimal signal-to-noise ratio

  • Run single-color controls for accurate compensation

  • Include FMO controls to set proper gates for LMAN1-positive populations

• Special considerations for LMAN1:

  • Account for cell type-specific expression differences (e.g., ~95% of lung DCs vs. ~15% of T cells)

  • Consider separate panels for analyzing surface versus intracellular LMAN1

  • Include markers to identify HDM-binding capability, as this correlates with LMAN1 expression