VEGFC Human, Sf9

How does the Sf9 expression system affect the structural properties of recombinant VEGFC?

The Sf9 baculovirus expression system produces a recombinant VEGFC that retains most of the structural characteristics of native VEGFC but with some important differences. The insect cell-based system provides post-translational modifications including glycosylation, though the glycosylation pattern differs from that of mammalian cells .

The Sf9-expressed VEGFC forms primarily non-covalently linked dimers, which is essential for its biological activity . This expression system yields protein with greater than 90% purity as determined by SDS-PAGE . The Sf9 system enables production of the functionally critical VEGF homology domain while excluding the N- and C-terminal extensions found in the pre-pro-protein, resulting in a protein that maintains receptor binding capabilities while being more manageable for research applications.

When designing experiments, researchers should consider that while the core structure is preserved, subtle differences in glycosylation patterns between Sf9-produced and mammalian-produced VEGFC might affect certain aspects of protein-protein interactions and cellular responses.

What are the optimal storage conditions for maintaining VEGFC stability and activity?

Proper storage of VEGFC Human, Sf9 is critical for maintaining its biological activity. The recommended storage protocols are as follows:

• For short-term use (2-4 weeks): Store at 4°C if the entire vial will be used within this timeframe .

• For medium-term storage: Store frozen at -20°C .

• For long-term storage: Store at -20°C with the addition of a carrier protein (0.1% Human Serum Albumin or Bovine Serum Albumin) .

It is crucial to avoid multiple freeze-thaw cycles as these can significantly reduce protein activity . The typical formulation of VEGFC protein solution (0.5 mg/ml) contains Phosphate Buffered Saline (pH 7.4) and 20% glycerol, which helps stabilize the protein during freeze-thaw processes .

What methodology should researchers employ when reconstituting and diluting VEGFC for experimental use?

When working with VEGFC Human, Sf9, proper reconstitution and dilution techniques are essential for maintaining protein activity:

• If receiving a lyophilized form, reconstitute using sterile PBS or the recommended buffer specified by the manufacturer.

• Allow the protein to fully dissolve by gentle swirling and avoid vortexing, which can cause protein denaturation.

• For dilutions, use buffers containing carrier proteins (0.1% BSA or HSA) to prevent protein loss through adsorption to labware surfaces .

• Prepare working dilutions immediately before use and avoid storing diluted solutions.

• When diluting, maintain a minimum concentration of 10 μg/ml to prevent significant activity loss.

For accurate activity assessment, it's recommended to use freshly reconstituted or thawed protein, as activity can decrease with storage time even under optimal conditions. When designing dose-response experiments, prepare a fresh dilution series for each experiment rather than storing pre-diluted solutions.

Which receptors does VEGFC interact with and what are their binding kinetics?

VEGFC is known to interact with two primary receptors of the tyrosine kinase family:

• VEGFR-3/FLT-4: This is the primary receptor for VEGFC and is strongly expressed in lymphatic endothelial cells. The affinity constant (Kd) for VEGFC binding to VEGFR-3 has been determined to be approximately 0.29 nM using Surface Plasmon Resonance (SPR) assays . In ELISA assays, the EC50 for VEGFC binding to VEGFR-3 is approximately 19.8 ng/ml .

• VEGFR-2/KDR: VEGFC can also bind to VEGFR-2, which is predominantly expressed on vascular endothelial cells, though with lower affinity than to VEGFR-3 .

The binding characteristics of VEGFC to these receptors are critical for its biological functions. The dual receptor specificity explains VEGFC's ability to influence both lymphatic vessel development (primarily via VEGFR-3) and, to a lesser extent, blood vessel angiogenesis (via VEGFR-2). This dual specificity should be considered when designing experiments to discriminate between effects on lymphatic versus blood vascular systems.

How can researchers effectively design experiments to evaluate VEGFC-mediated signaling pathways?

To effectively study VEGFC-mediated signaling, researchers should consider the following methodological approaches:

• Receptor-specific assays: Design experiments using cells expressing either VEGFR-2 or VEGFR-3 exclusively to distinguish receptor-specific responses.

• Phosphorylation analysis: Monitor receptor tyrosine phosphorylation and downstream signaling components such as Akt, ERK1/2, and PLCγ using phospho-specific antibodies in Western blot analyses.

• Dose-response studies: Establish dose-response relationships by treating cells with increasing concentrations of VEGFC (typically ranging from 1-100 ng/ml), based on the established EC50 of approximately 19.8 ng/ml for VEGFR-3 binding .

• Time-course experiments: Include multiple time points (5 min to 24 hours) to capture both immediate signaling events and delayed transcriptional responses.

• Receptor blocking: Use receptor-specific blocking antibodies or soluble receptor domains to confirm signaling specificity.

• Comparative analysis: Compare responses to VEGFC with those to other VEGF family members to identify pathway overlaps and distinctions.

When interpreting results, consider that the recombinant VEGFC from Sf9 cells forms primarily non-covalently linked dimers, which is the active configuration for receptor binding and signaling initiation .

What are the validated functional assays for assessing VEGFC activity in lymphangiogenesis research?

Several established assays can be employed to assess the biological activity of VEGFC Human, Sf9 in lymphangiogenesis research:

• Lymphatic Endothelial Cell (LEC) Proliferation Assay: Measure proliferation of primary LECs or immortalized LEC lines (e.g., hTERT-HDLECs) following VEGFC treatment using BrdU incorporation, Ki67 staining, or real-time cell analysis.

• Tube Formation Assay: Assess the ability of LECs to form capillary-like structures when cultured on Matrigel or other extracellular matrix substitutes in the presence of VEGFC.

• Migration Assays: Evaluate LEC migration using wound healing (scratch) assays or Boyden chamber/transwell migration assays with VEGFC as a chemoattractant.

• Spheroid Sprouting Assay: Generate LEC spheroids and embed them in collagen matrices to assess 3D sprouting responses to VEGFC gradients.

• Ex Vivo Lymphatic Ring Assay: Culture lymphatic vessel explants (e.g., from thoracic ducts) in 3D matrices and quantify lymphatic sprouting in response to VEGFC.

• VEGFR-3 Phosphorylation: Directly measure VEGFR-3 phosphorylation levels after VEGFC treatment using phospho-specific antibodies in Western blotting or ELISA formats.

For rigorous validation, it's recommended to include positive controls (e.g., full-length VEGFC or VEGF-D) and negative controls (e.g., heat-inactivated VEGFC or unrelated growth factors). Additionally, receptor specificity can be confirmed using VEGFR-3 blocking antibodies or soluble receptor domains.

How should researchers design experiments to differentiate between VEGFC effects on lymphatic versus blood vascular systems?

Designing experiments to distinguish between VEGFC's effects on lymphatic versus blood vascular systems requires careful consideration of cell types, markers, and experimental approaches:

• Cell-type selection: Use well-characterized lymphatic endothelial cells (LECs) and blood vessel endothelial cells (BECs) in parallel experiments. Human dermal lymphatic endothelial cells (HDLECs) for lymphatic studies and human umbilical vein endothelial cells (HUVECs) for vascular studies represent well-established models.

• Marker analysis: Examine lymphatic-specific markers (LYVE-1, Podoplanin, Prox-1) versus blood vessel markers (CD31high/Podoplanin-negative, von Willebrand Factor) to confirm cell identity and responses.

• Dose-response comparisons: Since VEGFC has higher affinity for VEGFR-3 than VEGFR-2, conduct dose-response studies across a wide concentration range (1-500 ng/ml) to identify differential sensitivity thresholds.

• Receptor blocking experiments: Use receptor-specific blocking antibodies against VEGFR-2 or VEGFR-3 to determine which receptor mediates observed effects.

• Co-culture systems: Develop co-culture models containing both LECs and BECs to observe competitive effects when both cell types are simultaneously exposed to VEGFC.

• 3D models: Employ 3D models such as lymphatic/blood vessel organoids or ex vivo explant cultures to assess effects in more physiologically relevant systems.

• In vivo models: Consider using transgenic reporter mice with fluorescently labeled lymphatic and blood vessels for in vivo studies following VEGFC administration.

When interpreting results, remember that while VEGFC primarily targets lymphatic endothelium through VEGFR-3, effects on blood vessels via VEGFR-2 may occur at higher concentrations or under specific experimental conditions .

How can researchers effectively use VEGFC Human, Sf9 in cancer research models?

VEGFC plays significant roles in tumor lymphangiogenesis and metastasis, making it a valuable research tool in cancer studies. When utilizing VEGFC Human, Sf9 in cancer research models, consider these methodological approaches:

• Tumor Lymphangiogenesis Assays:

  • Treat tumor cell lines with VEGFC to assess paracrine effects on co-cultured LECs

  • Create VEGFC-overexpressing tumor cell lines using the amino acid sequence information provided to study effects on tumor progression

  • Utilize 3D tumor spheroid-LEC co-culture systems to model tumor-lymphatic interactions

• Metastasis Models:

  • Apply VEGFC in orthotopic tumor models to evaluate effects on lymphatic vessel density and functionality

  • Assess sentinel lymph node changes following VEGFC exposure in animal models

  • Develop in vitro models of tumor cell transendothelial migration across LEC monolayers in response to VEGFC

• Therapeutic Targeting Studies:

  • Use VEGFC as a positive control when testing VEGFR-3 inhibitors or VEGFC-neutralizing antibodies

  • Employ VEGFC to establish dose-response relationships for anti-lymphangiogenic compounds

  • Develop reporter assays incorporating VEGFC-VEGFR3 signaling for high-throughput drug screening

• Biomarker Research:

  • Correlate experimental VEGFC levels with lymphatic vessel density and metastatic potential

  • Establish standard curves using recombinant VEGFC for quantitative assessment of VEGFC in clinical samples

When designing these experiments, consider that VEGFC from Sf9 cells forms primarily non-covalently linked dimers, which is the active configuration for receptor signaling . For optimal activity, adhere to the recommended storage conditions and avoid multiple freeze-thaw cycles .

What methodologies are recommended for studying VEGFC processing and regulation?

VEGFC undergoes complex processing that regulates its bioavailability and activity. When studying VEGFC processing and regulation, researchers should consider these methodological approaches:

• Proteolytic Processing Analysis:

  • Use Western blotting to track the different forms of VEGFC (pre-pro-protein of 416 amino acids, partially processed forms, and mature form)

  • Design experiments comparing the recombinant VEGFC (aa 112-227) to full-length VEGFC to understand functional differences

  • Employ protease inhibitors to identify key enzymes involved in VEGFC maturation

• Gene Expression Regulation:

  • Analyze tissue-specific expression patterns, noting that in adults, VEGFC is highly expressed in heart, placenta, ovary, and small intestine

  • Design reporter assays using VEGFC promoter regions to study transcriptional regulation

  • Utilize qPCR and RNA-seq to examine VEGFC expression under different physiological and pathological conditions

• Post-translational Modification Studies:

  • Compare glycosylation patterns between Sf9-produced VEGFC and mammalian-produced VEGFC

  • Assess the impact of glycosylation on receptor binding using deglycosylated VEGFC variants

  • Examine the effects of other post-translational modifications on VEGFC activity

• Protein-Protein Interaction Analysis:

  • Investigate VEGFC interactions with extracellular matrix components using the His-tagged recombinant protein

  • Study co-receptor interactions that might modulate VEGFC activity

  • Employ co-immunoprecipitation studies to identify novel VEGFC-interacting proteins

• Binding Kinetics Assessment:

  • Use Surface Plasmon Resonance (SPR) to assess binding kinetics, referencing the established affinity constant of 0.29 nM for VEGFR-3

  • Develop competitive binding assays to study receptor specificity

When designing these experiments, researchers should consider that the recombinant VEGFC from Sf9 represents only the middle VEGF homology domain of the full protein and lacks the N- and C-terminal extensions present in the native pre-pro-protein .

What are common challenges when working with VEGFC Human, Sf9 and how can researchers overcome them?

Researchers often encounter several challenges when working with VEGFC Human, Sf9. Here are common issues and recommended solutions:

• Activity Loss During Storage:

  • Problem: Decreased biological activity after storage

  • Solution: Store at 4°C if using within 2-4 weeks; for longer periods, store at -20°C with addition of carrier protein (0.1% HSA or BSA) ; aliquot protein solutions to avoid multiple freeze-thaw cycles

• Protein Adsorption to Labware:

  • Problem: Loss of protein concentration due to adsorption to tubes and pipette tips

  • Solution: Use low-binding tubes and tips; add carrier protein (0.1% BSA) to dilution buffers; pre-coat containers with BSA solution before adding VEGFC

• Inconsistent Bioactivity Results:

  • Problem: Variable responses in cell-based assays

  • Solution: Standardize cell passage numbers; ensure consistent cell density; conduct dose-response curves with each new lot of VEGFC; reference the established EC50 value of 19.8 ng/ml for VEGFR-3 binding

• Dimeric Structure Destabilization:

  • Problem: Loss of active dimeric configuration

  • Solution: Avoid harsh mixing methods like vortexing; use gentle rotation or pipetting for mixing; maintain appropriate buffer conditions (pH 7.4)

• Interference in Detection Assays:

  • Problem: His-tag interference with antibody binding or functional assays

  • Solution: Include appropriate controls; consider enzymatic removal of the tag if necessary; use detection antibodies that recognize epitopes distant from the tag region

• Glycosylation Heterogeneity:

  • Problem: Batch-to-batch variation in glycosylation patterns

  • Solution: Assess each lot by SDS-PAGE; note that molecular size on SDS-PAGE will appear at approximately 18-28 kDa due to glycosylation ; consider deglycosylation for applications requiring homogeneous protein

How can researchers validate the quality and activity of their VEGFC preparations before use in critical experiments?

Proper validation of VEGFC Human, Sf9 quality and activity is crucial before use in significant experiments. A comprehensive validation approach should include:

• Physical Characterization:

  • Purity assessment by SDS-PAGE (should be >90%)

  • Western blot analysis using anti-VEGFC antibodies to confirm identity

  • Size exclusion chromatography to verify dimeric state and absence of aggregates

• Functional Validation:

  • ELISA-based binding assay to VEGFR-3, comparing to the established EC50 of 19.8 ng/ml

  • Surface Plasmon Resonance (SPR) to confirm binding kinetics (reference affinity constant: 0.29 nM)

  • Phosphorylation assay of VEGFR-3 in responsive cells (e.g., HDLECs)

• Biological Activity Testing:

  • Proliferation assay using lymphatic endothelial cells

  • Migration assay (Boyden chamber or scratch assay)

  • Sprouting assay using 3D spheroid cultures of LECs

• Comparative Analysis:

  • Side-by-side comparison with a reference standard or previous lot

  • Dose-response curve generation to establish potency

  • Determination of specific activity (units of activity per mg protein)

• Stability Assessment:

  • Activity testing after storage under recommended conditions

  • Freeze-thaw stability analysis

  • Thermal stability evaluation

A minimum validation protocol should include at least one test from each category above. For critical experiments, consider developing a Certificate of Analysis that includes purity, binding activity (EC50), and biological functionality metrics for each batch of VEGFC used.

How does VEGFC from Sf9 cells compare to VEGFC expressed in other systems for research applications?

Different expression systems produce VEGFC with varying characteristics that can impact research applications. Here's a comparative analysis of VEGFC from Sf9 versus other expression systems:

For research applications:

• Basic Binding Studies: Sf9-expressed VEGFC is generally suitable for receptor binding assays, as evidenced by its demonstrated affinity to VEGFR-3 (EC50 of 19.8 ng/ml, Kd of 0.29 nM) .

• Cell-Based Functional Assays: For critical cell-based assays, especially with mammalian cells, HEK293-expressed VEGFC may provide more physiologically relevant responses due to human-type glycosylation.

• Structural Studies: For crystallography or structural analyses, E. coli-expressed non-glycosylated VEGFC might be preferred for homogeneity.

• In Vivo Applications: For animal studies, mammalian-expressed VEGFC typically offers better pharmacokinetics and reduced immunogenicity.

The choice of expression system should be guided by the specific research question, with consideration of the importance of glycosylation and other post-translational modifications to the particular application.

What experimental design considerations are critical when studying VEGFC interactions with VEGFR-3 versus VEGFR-2?

When investigating VEGFC interactions with its receptors, careful experimental design is essential to distinguish between VEGFR-3 and VEGFR-2 mediated effects:

• Receptor Expression Profiling:

  • Begin experiments by characterizing receptor expression levels in your cellular models

  • Quantify VEGFR-2 and VEGFR-3 expression using qPCR, Western blotting, or flow cytometry

  • Select appropriate positive control cell lines (e.g., HDLECs for VEGFR-3, HUVECs for VEGFR-2)

• Binding Kinetics Differentiation:

  • Design dose-response experiments accounting for different affinities (VEGFC binds VEGFR-3 with higher affinity than VEGFR-2)

  • Use the established EC50 of 19.8 ng/ml and affinity constant of 0.29 nM for VEGFR-3 as reference points

  • Employ competition assays with receptor-specific ligands (e.g., VEGF-A for VEGFR-2)

• Receptor-Specific Signaling Analysis:

  • Map downstream signaling pathways associated with each receptor

  • Design time-course experiments to capture both immediate (minutes) and delayed (hours) signaling events

  • Use receptor-specific inhibitors or blocking antibodies to confirm pathway specificity

• Receptor Knockout/Knockdown Approaches:

  • Employ CRISPR-Cas9 or siRNA techniques to selectively remove one receptor

  • Create isogenic cell lines differing only in receptor expression

  • Use receptor-negative cell lines transfected with either VEGFR-2 or VEGFR-3

• Mutational Analysis:

  • Consider using VEGFC mutants with altered receptor specificity

  • Reference the amino acid sequence provided to design or select appropriate mutants

• Physiological Relevance Assessment:

  • Design experiments to link receptor binding to functional outcomes

  • For VEGFR-3: focus on lymphatic endothelial cell proliferation, migration, and tube formation

  • For VEGFR-2: assess effects on blood vessel endothelial cells

• Quantitative Data Analysis:

  • Use appropriate statistical methods to distinguish specific from non-specific effects

  • Establish clear threshold criteria for positive responses

  • Consider advanced computational methods for pathway analysis

When interpreting results, remember that VEGFC primarily functions through VEGFR-3 in lymphatic endothelial cells, but at higher concentrations may also activate VEGFR-2, particularly in experimental settings with overexpressed receptors .

What emerging techniques can enhance VEGFC-related research beyond current methodologies?

Emerging technologies are expanding the possibilities for VEGFC research beyond traditional approaches. Researchers should consider these advanced methodologies:

• Single-Cell Analysis Technologies:

  • Apply single-cell RNA-seq to identify heterogeneous responses to VEGFC within lymphatic endothelial cell populations

  • Employ CyTOF or spectral flow cytometry to simultaneously analyze multiple signaling pathways activated by VEGFC

  • Utilize single-cell proteomics to identify differential protein expression patterns in response to VEGFC

• Advanced Imaging Techniques:

  • Implement light sheet microscopy for 3D visualization of VEGFC-induced lymphangiogenesis in organoids

  • Apply super-resolution microscopy (STORM, PALM) to study VEGFC-receptor clustering and internalization

  • Use intravital microscopy with fluorescently labeled VEGFC to track its distribution and effects in vivo

• Organ-on-Chip and Microfluidic Systems:

  • Develop lymphatic vessel-on-chip models to study VEGFC effects under physiologically relevant flow conditions

  • Create tumor-lymphatic interface chips to investigate VEGFC's role in metastasis

  • Employ gradient-generating microfluidic devices to assess directional migration responses to VEGFC

• CRISPR-Based Technologies:

  • Use CRISPR activation/interference systems to modulate VEGFC and receptor expression with temporal precision

  • Apply CRISPR screens to identify novel components of VEGFC signaling pathways

  • Implement base editing to introduce specific mutations in VEGFC or its receptors

• Computational and Systems Biology Approaches:

  • Develop mathematical models of VEGFC signaling networks using binding kinetics data (EC50 of 19.8 ng/ml, Kd of 0.29 nM)

  • Apply machine learning to predict cellular responses to VEGFC under various conditions

  • Use systems biology approaches to integrate multi-omics data related to VEGFC signaling

These emerging techniques can provide unprecedented insights into VEGFC biology beyond what is possible with traditional biochemical and cell biology approaches, potentially revealing new therapeutic targets and biological mechanisms.

How can multi-omics approaches be integrated to advance understanding of VEGFC signaling networks?

Integrating multi-omics approaches provides a comprehensive view of VEGFC signaling networks and their biological consequences. Here's a methodological framework for such integration:

• Coordinated Experimental Design:

  • Plan experiments that generate matched samples for multiple omics analyses

  • Design time-course studies (5 min to 24h post-VEGFC exposure) to capture dynamic changes

  • Include appropriate controls (untreated, heat-inactivated VEGFC, receptor-blocking conditions)

• Transcriptomic Analysis:

  • Perform RNA-seq to identify VEGFC-regulated genes

  • Use nascent RNA sequencing to distinguish primary from secondary transcriptional responses

  • Apply single-cell RNA-seq to identify cell subpopulation-specific responses

• Proteomic and Phosphoproteomic Integration:

  • Conduct global proteomics to identify changes in protein abundance

  • Perform phosphoproteomics focusing on early time points (5-30 min) to map signaling cascade activation

  • Use proximity labeling techniques to identify VEGFC receptor interactomes

• Metabolomic Analysis:

  • Profile metabolic changes in VEGFC-stimulated cells

  • Focus on glycolysis, fatty acid metabolism, and other pathways critical for endothelial cell function

  • Correlate metabolic shifts with functional outcomes like proliferation and migration

• Chromatin Accessibility and Epigenetic Studies:

  • Apply ATAC-seq to identify chromatin regions that become accessible after VEGFC treatment

  • Use ChIP-seq for key transcription factors downstream of VEGFC signaling

  • Integrate with transcriptomic data to build gene regulatory networks

• Data Integration Methodologies:

  • Employ computational tools to integrate multi-omics datasets (e.g., WGCNA, MOFA)

  • Apply network analysis to identify key nodes in VEGFC signaling

  • Use Bayesian approaches to infer causal relationships between molecular events

• Validation Strategies:

  • Verify key findings using targeted approaches (qPCR, Western blotting)

  • Apply CRISPR-based perturbations to validate predicted network connections

  • Correlate molecular changes with functional outcomes in lymphatic endothelial cells

This integrated approach can reveal previously unrecognized components of VEGFC signaling networks, providing a systems-level understanding of how this growth factor orchestrates complex cellular responses, particularly in the context of lymphatic vessel development and function.