LYVE1 Mouse Sf9

What is LYVE1 Mouse Sf9 and what is its biological significance?

LYVE1 Mouse Sf9 refers to recombinant mouse Lymphatic Vessel Endothelial Hyaluronic Acid Receptor 1 produced in Sf9 insect cells. This soluble protein represents a key receptor for hyaluronic acid (HA) on lymph vessel walls. Biologically, LYVE1 serves as the first characterized lymph-specific HA receptor and functions as a uniquely powerful marker for lymphatic vessels themselves .

The recombinant protein contains the extracellular domain fused to a C-terminal His-tag (6xHis) produced in baculovirus. It is a monomeric, glycosylated polypeptide containing 228 amino acids (Met-1 to Gly 228) with a core molecular mass of 25 kDa, though glycosylation increases the apparent molecular weight to approximately 40 kDa .

LYVE1's biological significance extends beyond being merely a marker. It functions as a ligand-specific transporter trafficking between intracellular organelles (trans-Golgi network) and the plasma membrane. Current evidence suggests it plays roles in:

• Autocrine regulation of cell growth

• Hyaluronan transport and catabolism

• Lymphatic vessel development and function

• Cell adhesion and signal transduction processes

How does LYVE1 differ from other hyaluronic acid receptors like CD44?

While LYVE1 shares approximately 41% sequence similarity with CD44 (another important HA receptor), several key differences make LYVE1 particularly valuable for lymphatic research:

This specific expression pattern makes LYVE1 invaluable for distinguishing lymphatic vessels from blood vessels in research contexts, particularly in studies of lymphangiogenesis, lymphatic metastasis, and lymphedema.

What are the optimal storage and handling conditions for LYVE1 Mouse Sf9?

Proper storage and handling of LYVE1 Mouse Sf9 are essential for maintaining its stability and activity. Manufacturer recommendations include:

For lyophilized protein:

• Store desiccated below -18°C for long-term stability

• While stable at room temperature for up to 3 weeks, refrigerated or frozen storage is preferred

After reconstitution:

• Store at 4°C if using within 2-7 days

• For longer storage, maintain below -18°C

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Consider adding carrier protein (0.1% HSA or BSA) for long-term storage

Reconstitution protocol:

• Reconstitute lyophilized LYVE1 in sterile water to achieve concentration ≥100 μg/ml

• Allow complete solubilization before further dilution

• The solution can then be diluted into other aqueous buffers as needed for specific applications

If small volumes become entrapped in the vial seal during shipment or storage, briefly centrifuge the vial to collect all material before reconstitution .

What reconstitution protocols ensure optimal LYVE1 activity for functional studies?

For functional studies requiring active LYVE1, the following reconstitution protocol is recommended:

Standard Reconstitution Protocol:

• Allow lyophilized protein to equilibrate to room temperature (approximately 15-30 minutes)

• Briefly centrifuge the vial to collect all material

• Add sterile water directly to achieve concentration ≥100 μg/ml

• Gently mix by rotating or mild vortexing, avoiding excessive agitation

• Allow complete dissolution (10-15 minutes) before proceeding

For specific application types:

For binding assays:

• Consider reconstituting in a buffer compatible with downstream applications (e.g., PBS pH 7.2-7.4)

• Add 0.1% BSA to prevent non-specific binding and surface adsorption

• For hyaluronic acid binding studies, include 1-2 mM calcium and magnesium ions

For cell culture experiments:

• After initial reconstitution, dilute in appropriate cell culture medium

• Filter through 0.22 μm filter if sterility is critical

• Determine optimal working concentration through titration experiments

Quality control after reconstitution:

• Verify protein concentration using appropriate methods (Bradford, BCA, etc.)

• Assess binding activity with a simple HA binding assay

• Check for aggregation using UV-Vis spectroscopy or dynamic light scattering

How can researchers validate LYVE1 activity after reconstitution?

Validating LYVE1 activity after reconstitution is critical for experimental reliability. Several complementary approaches should be employed:

• Hyaluronic acid (HA) binding assay:

  • Coat plates with high molecular weight HA (100 μg/ml)

  • Block with 1-2% BSA

  • Add reconstituted LYVE1 at various concentrations

  • Detect bound LYVE1 using anti-His antibody or specific anti-LYVE1 antibody

  • Compare binding curve to reference standards

  • A dose-dependent binding curve indicates functional protein

• Structural integrity assessment:

  • SDS-PAGE under reducing and non-reducing conditions

  • Western blot using conformation-specific antibodies

  • Size-exclusion chromatography to confirm monomeric state and absence of aggregation

• Functional cell-based validation:

  • Test binding to lymphatic endothelial cells expressing hyaluronic acid

  • Compare activity with previously validated LYVE1 preparations

  • Implement positive and negative controls to establish assay dynamic range

If activity appears compromised, consider:

• Optimizing buffer conditions (pH, ionic strength, divalent cations)

• Adding stabilizing agents (glycerol, carrier proteins)

• Preparing fresh reconstitution if protein has been stored for extended periods

• Checking for potential aggregation or precipitation

What experimental approaches can be used to study LYVE1's role in lymphangiogenesis?

Several methodological approaches allow researchers to investigate LYVE1's role in lymphangiogenesis:

• Immunohistochemistry/Immunofluorescence:

  • Use anti-LYVE1 antibodies to identify lymphatic vessels in tissue sections

  • Co-stain with other lymphatic markers (Prox1, podoplanin) for confirmation

  • Compare with blood vessel markers (CD31) to distinguish vessel types

  • Quantify lymphatic vessel density, size, and branching patterns

  • Track temporal expression during development or pathological processes

• Functional assays:

  • Tube formation assays with lymphatic endothelial cells

  • Lymphatic endothelial cell migration assays

  • Examine effects of LYVE1 blocking or stimulation

  • Correlate LYVE1 expression with hyaluronic acid transport

• Gene expression manipulation:

  • siRNA knockdown of LYVE1 in lymphatic endothelial cells

  • Overexpression studies to assess gain-of-function effects

  • CRISPR/Cas9 modification for precise genetic alterations

  • Compare with VEGF-C expression patterns, noting LYVE1 expression typically follows VEGF-C with a 1-2 day delay

• In vivo models:

  • Transgenic mouse models with LYVE1 manipulation

  • Implantation of LYVE1-modified cells

  • Examination of lymphatic development in embryoid bodies

  • Real-time imaging of lymphatic vessel formation using LYVE1 as a marker

These approaches can be combined to provide comprehensive insights into LYVE1's functional roles in lymphangiogenesis, from molecular interactions to systemic effects.

How can researchers address potential discrepancies in LYVE1 expression data?

Researchers often encounter discrepancies in LYVE1 expression patterns across different experimental systems. A systematic approach to addressing these includes:

• Methodological standardization:

  • Use multiple antibody clones targeting different LYVE1 epitopes

  • Apply consistent detection protocols across experiments

  • Standardize image analysis parameters and thresholds

  • Employ both protein and mRNA detection methods when possible

• Multi-marker approach:

  • Always use multiple lymphatic markers alongside LYVE1

  • Consider hierarchical marker combinations (LYVE1+/Prox1+/podoplanin+)

  • Distinguish lymphatic endothelial cells from other LYVE1+ populations (like certain macrophages)

• Biological context considerations:

  • Document developmental stage-specific patterns

  • Consider species differences in expression patterns

  • Account for tissue-specific regulatory mechanisms

  • Note pathological condition effects (inflammation, tumor microenvironment)

• Validation strategies:

  • Verify findings using orthogonal techniques

  • Compare results with published literature systematically

  • Collaborate with other laboratories for independent confirmation

  • Consider single-cell approaches to resolve heterogeneity issues

How do glycosylation patterns of Sf9-produced LYVE1 affect its functionality?

The glycosylation pattern of LYVE1 produced in Sf9 insect cells represents an important consideration for advanced research applications. These patterns differ significantly from those in mammalian systems:

• Sf9 cell glycosylation characteristics:

  • Primarily produce paucimannose N-glycans

  • Lack complex mammalian-type glycans with terminal sialic acid

  • Often show reduced O-linked glycosylation

  • The apparent molecular weight of 40 kDa (versus 25 kDa core protein) confirms glycosylation occurs, but with different patterns

• Functional implications:

  • The Link module domain generally maintains HA binding activity despite altered glycosylation

  • Binding affinity may differ quantitatively from mammalian-expressed LYVE1

  • Receptor clustering and multimerization properties may be affected

  • Interactions with glycan-binding proteins could be modified

• Methodological approaches to address this:

  • Include mammalian-expressed LYVE1 controls when possible

  • Use comparative binding assays to quantify any affinity differences

  • Consider enzymatic deglycosylation to assess glycan contribution to function

  • For structural studies, Sf9-produced protein remains valuable regardless of glycosylation differences

What molecular interactions mediate LYVE1's function in lymphatic vessels?

LYVE1 participates in a complex network of molecular interactions within lymphatic vessels, which advanced research continues to elucidate:

• Primary hyaluronan (HA) interactions:

  • LYVE1 binds both soluble and immobilized HA through its Link module domain

  • This interaction facilitates HA uptake and transport within lymphatic vessels

  • Different HA fragment sizes may trigger distinct LYVE1-mediated responses

  • LYVE1 may form clusters upon HA binding, enhancing avidity

• Signaling pathway interactions:

  • VEGF-C/VEGFR-3: LYVE1 expression patterns closely follow VEGF-C, suggesting coordinated regulation in lymphangiogenesis

  • Expression timing evidence indicates LYVE1 peaks 1-2 days after VEGF-C expression

  • LYVE1 may function within autocrine cell growth regulation pathways

  • Potential role in extracellular matrix remodeling during lymphatic development

• Cell-cell interaction mediators:

  • Co-expression with other lymphatic markers like podoplanin suggests functional integration

  • May interact with Prox1-regulated pathways for lymphatic endothelial cell differentiation

  • Potential role in immune cell trafficking through lymphatic vessels

• Transport function:

  • Functions as a hyaluronan transporter in lymphatic endothelial cells

  • May mediate HA uptake for catabolism within lymphatic endothelial cells

  • Alternatively facilitates HA transport into afferent lymphatic vessels for subsequent degradation in lymph nodes

These molecular interactions position LYVE1 as both a marker and functional component in lymphatic biology, with emerging roles beyond simple HA binding that continue to be elucidated through advanced research methodologies.

What quality control measures ensure experimental reproducibility when using LYVE1 Mouse Sf9?

Ensuring reproducibility with LYVE1 Mouse Sf9 requires comprehensive quality control measures throughout the research workflow:

• Initial protein characterization:

  • Verify purity by SDS-PAGE (should exceed 95% as determined by RP-HPLC and SDS-PAGE)

  • Confirm identity by Western blot with anti-LYVE1 and anti-His antibodies

  • Validate molecular weight (approximately 40 kDa due to glycosylation)

  • Document batch information and certificate of analysis

• Functional validation:

  • Perform HA binding assay with each new lot

  • Compare binding curves to established reference standards

  • Establish acceptance criteria for experimental use

  • Test functionality in relevant cell-based assays when applicable

• Standardized protocols:

  • Develop detailed SOPs for reconstitution and storage

  • Implement consistent experimental workflows

  • Document all protocol deviations and lot numbers

  • Maintain electronic records of all experimental parameters

• Appropriate controls:

  • Include positive controls (validated previous lots)

  • Use negative controls (non-LYVE1 proteins with similar properties)

  • Implement vehicle/buffer controls

  • Consider internal reference samples across experiments

• Advanced validation:

  • Perform orthogonal method verification of key findings

  • Consider inter-laboratory validation for critical results

  • Use automated systems where possible to reduce operator variability

  • Implement statistical approaches appropriate for the experimental design

By systematically implementing these quality control measures, researchers can significantly enhance experimental reproducibility when working with LYVE1 Mouse Sf9, leading to more reliable and translatable research findings.

What are the emerging advanced techniques for studying LYVE1 in lymphatic research?

Lymphatic research continues to evolve with cutting-edge methodological approaches that enhance our understanding of LYVE1's role:

• Single-cell technologies:

  • Single-cell RNA sequencing reveals heterogeneity in LYVE1 expression among lymphatic endothelial cells

  • Mass cytometry (CyTOF) allows simultaneous measurement of LYVE1 with dozens of other markers

  • Spatial transcriptomics preserves tissue context while profiling LYVE1 expression patterns

• Advanced imaging techniques:

  • Light sheet microscopy enables 3D visualization of entire lymphatic networks with LYVE1 labeling

  • Super-resolution microscopy resolves LYVE1 distribution at nanometer scale

  • Intravital microscopy tracks LYVE1+ vessel dynamics in living organisms

• Genetic and genome editing approaches:

  • CRISPR/Cas9-mediated LYVE1 modification creates precise knockouts or tagged variants

  • Conditional and inducible systems control LYVE1 expression with temporal and spatial precision

  • AAV-mediated gene transfer delivers modified LYVE1 constructs to specific tissues

• Systems biology integration:

  • Multi-omics approaches combine transcriptomics, proteomics, and metabolomics data

  • Network analysis places LYVE1 within broader lymphatic development pathways

  • Mathematical modeling predicts effects of LYVE1 perturbation on lymphatic development

These advanced techniques are pushing the boundaries of LYVE1 research, moving beyond descriptive studies to mechanistic understanding of complex lymphatic development and function in both normal and pathological conditions.

What are common challenges when working with LYVE1 Mouse Sf9 and how can they be addressed?

Researchers frequently encounter several challenges when working with LYVE1 Mouse Sf9 that require systematic troubleshooting:

• Protein stability and aggregation issues:

  • Challenge: LYVE1 may form aggregates during storage or after reconstitution

  • Solution: Add carrier proteins (0.1% BSA) to reconstitution buffer, use stabilizing agents like glycerol (10%), and filter through 0.22 μm filter after reconstitution

• Variable binding activity:

  • Challenge: Inconsistent hyaluronic acid binding between experiments

  • Solution: Standardize buffer conditions (include divalent cations), use high molecular weight HA for binding studies, and establish internal reference standards for batch comparison

• Reconstitution difficulties:

  • Challenge: Poor solubility or precipitation after reconstitution

  • Solution: Ensure reconstitution at recommended concentration (≥100 μg/ml), reconstitute slowly at 4°C rather than room temperature, and centrifuge briefly before use to remove any particulates

• Storage-related activity loss:

  • Challenge: Diminished activity after storage periods

  • Solution: Store desiccated below -18°C when lyophilized, prepare single-use aliquots after reconstitution, add carrier protein for long-term storage of reconstituted protein, and avoid repeated freeze-thaw cycles

• Detection challenges in complex samples:

  • Challenge: Difficulty detecting LYVE1 in tissue samples or cell cultures

  • Solution: Optimize antibody concentrations, use multiple antibody clones recognizing different epitopes, include positive controls, and consider antigen retrieval methods for fixed tissues

By anticipating these challenges and implementing appropriate solutions, researchers can significantly improve the reliability and reproducibility of experiments using LYVE1 Mouse Sf9.

How can researchers optimize buffer conditions for LYVE1 functional assays?

Optimizing buffer conditions is critical for maintaining LYVE1 activity in functional assays. The following parameters should be considered:

• Buffer composition for HA binding assays:

  • Base buffer: PBS (pH 7.2-7.4) provides physiological conditions

  • Divalent cations: Include 1-2 mM calcium and magnesium to enhance binding

  • Protein stabilizers: Add 0.1-0.5% BSA to prevent non-specific binding and protein adsorption

  • Detergents: Low concentrations (0.01-0.05% Tween-20) may reduce background, but higher concentrations can disrupt HA interactions

• pH optimization:

  • Optimal range: pH 7.2-7.4 typically provides best activity

  • Stability testing: If studying pH-dependent effects, test stability at each pH value

  • Buffer systems: Phosphate buffers work well but consider HEPES (20 mM) for better pH stability

• Ionic strength considerations:

  • Standard condition: 150 mM NaCl mimics physiological conditions

  • High salt effect: Increasing to 300 mM NaCl can reduce non-specific interactions

  • Low salt effect: Reducing to 50 mM may enhance some electrostatic interactions

• Temperature factors:

  • Standard assays: Room temperature (22-25°C) provides good activity

  • Kinetic studies: Compare binding at 4°C, 25°C, and 37°C

  • Storage: Keep reconstituted protein at 4°C for short-term use

• Protein concentration optimization:

  • Starting range: 0.1-10 μg/ml for most applications

  • Binding assays: Generate dose-response curves to determine optimal concentration

  • Cell-based assays: Titrate to find minimum effective concentration

Through systematic optimization of these buffer parameters, researchers can significantly enhance LYVE1 activity in functional assays, leading to more robust and reproducible experimental outcomes.

What future research directions are emerging for LYVE1 Mouse Sf9 applications?

The utility of LYVE1 Mouse Sf9 continues to expand as new research directions emerge in lymphatic biology. Several promising research frontiers include:

• Advanced lymphatic imaging applications:

  • Development of LYVE1-based molecular imaging probes for non-invasive lymphatic visualization

  • Integration with emerging imaging technologies for higher resolution lymphatic mapping

  • Quantitative approaches to assess lymphatic vessel functionality in development and disease

• Therapeutic targeting strategies:

  • Exploration of LYVE1 as a potential target for lymphedema treatments

  • Investigation of LYVE1-mediated drug delivery to lymphatic tissues

  • Development of anti-lymphangiogenic approaches targeting LYVE1 for cancer therapy

  • Engineering LYVE1-based constructs for immunomodulation

• Systems biology integration:

  • Placement of LYVE1 within comprehensive lymphatic development networks

  • Multi-omics approaches to understand LYVE1 regulation and function

  • Computational modeling of LYVE1 interactions within the lymphatic system

  • Integration of LYVE1 data with broader immunological and vascular biology

• Single-cell and spatial biology:

  • Higher-resolution understanding of LYVE1 expression heterogeneity

  • Mapping of spatial relationships between LYVE1+ structures and surrounding tissues

  • Temporal dynamics of LYVE1 expression during development and pathological processes

  • Identification of new LYVE1+ cell populations with distinct functional properties

These emerging directions highlight the continuing importance of LYVE1 Mouse Sf9 as a valuable tool for advancing our understanding of lymphatic biology in both normal physiology and disease states.