Endoglin Human, Sf9

What are the recommended storage and stability conditions for Endoglin Human, Sf9?

For long-term storage, lyophilized Endoglin Human, Sf9 should be stored desiccated below -18°C, although it remains stable at room temperature for up to 3 weeks. After reconstitution, the protein should be stored at 4°C if used within 2-7 days. For longer storage after reconstitution, it should be kept below -18°C. To enhance stability, adding a carrier protein (0.1% HSA or BSA) is recommended. Multiple freeze-thaw cycles should be avoided to prevent protein degradation .

What is the biological role of endoglin in normal physiology?

Endoglin plays crucial roles in multiple physiological processes. It functions as a co-receptor in the TGF-β signaling pathway, binding to TGF-β1, TGF-β3, activin-A, BMP-2, and BMP-7. Beyond TGF-β signaling, endoglin is involved in cytoskeletal organization affecting cell morphology and migration. It plays a vital role in cardiovascular system development and vascular remodeling. Experimental mice lacking the endoglin gene die due to cardiovascular abnormalities, highlighting its essential role in development .

How should Endoglin Human, Sf9 be reconstituted for experimental use?

The lyophilized Endoglin Human, Sf9 should be reconstituted in sterile PBS at a concentration not less than 100 μg/ml. For the standard version with carrier protein, reconstitution at 250 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin is recommended. For the carrier-free version, reconstitute at 250 μg/mL in sterile PBS without additional proteins. The reconstituted solution can then be further diluted to prepare working solutions for specific experimental applications .

How does endoglin modulate the TGF-β signaling pathway, and what experimental approaches can best elucidate these interactions?

Endoglin modulates TGF-β signaling by functioning as an accessory receptor that regulates the binding of various TGF-β family ligands. To investigate these interactions, researchers should consider using co-immunoprecipitation assays to detect protein-protein interactions between endoglin and TGF-β receptors, phosphorylation studies to assess SMAD activation, and reporter gene assays to measure downstream transcriptional activity.

Experimental evidence shows that endoglin regulates the TGF-β/SMAD3/VEGF signaling axis in cancer cells. For effective study design, combining genetic approaches (siRNA knockdown or CRISPR/Cas9) with recombinant protein treatments can help delineate the specific role of endoglin in modulating signaling outcomes. Immunofluorescence studies for SMAD3 nuclear translocation can provide visual evidence of pathway activation or inhibition .

What are the methodological considerations when using Endoglin Human, Sf9 in angiogenesis assays?

When designing angiogenesis assays with Endoglin Human, Sf9, several methodological considerations are crucial. For in vitro tube formation assays, HUVECs should be seeded on Matrigel-coated plates, with careful attention to cell density (approximately 1 × 10^4 cells per well in 24-well plates).

When evaluating the effect of endoglin on angiogenesis, transwell systems can be employed where test cells (e.g., control and experimental cancer cells) are seeded in 0.4 μm transwell inserts placed above the HUVEC cultures. Image capture should occur at standardized timepoints (typically 8 hours after seeding) to allow for proper tube formation. Analysis should use specialized software like WIMASIS Image Analysis for quantification of parameters including tube length, branch points, and loop formation .

Additionally, complementary assays such as VEGF ELISA of conditioned media should be performed to correlate angiogenic potential with growth factor production. For in vivo validation, consider zebrafish xenograft models, which allow for direct visualization of sprouting blood vessels in response to implanted cells .

How can researchers address the heterogeneity in glycosylation patterns when working with Endoglin Human, Sf9?

Glycosylation heterogeneity in Sf9-produced proteins presents a significant challenge for researchers. To address this:

• Characterize the glycosylation profile using mass spectrometry and lectin binding assays to establish baseline heterogeneity patterns.

• Consider enzymatic deglycosylation using PNGase F or Endo H, followed by functional assays to determine if glycosylation affects protein activity.

• For critical experiments requiring homogeneous preparations, implement additional purification steps such as lectin affinity chromatography to separate differently glycosylated isoforms.

• Include multiple batches of the recombinant protein in experimental designs to account for batch-to-batch variation in glycosylation patterns.

• When comparing experimental results across studies, explicitly document the apparent molecular weight observed in your SDS-PAGE analysis as an indicator of glycosylation status.

• For interaction studies, consider using both glycosylated and deglycosylated forms to determine if sugar moieties influence binding properties to TGF-β family ligands.

What experimental approaches can effectively demonstrate the functional differences between membrane-bound endoglin and the soluble recombinant form?

To investigate functional differences between membrane-bound endoglin and soluble recombinant forms:

• Design comparative binding assays using surface plasmon resonance (SPR) to measure affinity constants for various ligands (TGF-β1, TGF-β3, BMP-2, etc.) between the two forms.

• Perform cell-based signaling assays using reporter systems (e.g., SMAD-responsive luciferase constructs) to compare how each form modulates TGF-β pathway activation.

• Establish competitive binding experiments where soluble endoglin is added to cells expressing membrane-bound endoglin to assess potential dominant-negative effects.

• Use chemical crosslinking followed by immunoprecipitation to compare protein-protein interaction profiles between the two forms.

• Implement CRISPR/Cas9-mediated endoglin knockout cell lines complemented with either full-length membrane-bound endoglin or constitutively secreted soluble endoglin to compare cellular phenotypes.

• For angiogenesis research, compare tube formation assays where HUVECs are treated with either soluble recombinant endoglin or co-cultured with cells expressing membrane-bound endoglin .

What are the optimal conditions for using Endoglin Human, Sf9 in binding studies with TGF-β family ligands?

For optimal binding studies between Endoglin Human, Sf9 and TGF-β family ligands:

• Buffer composition: Use a physiological buffer system such as PBS (pH 7.2-7.4) supplemented with 0.1% BSA to reduce non-specific binding and prevent protein adsorption to surfaces.

• Temperature: Conduct binding assays at room temperature (25°C) or 37°C to mimic physiological conditions, with temperature consistency maintained throughout experiments.

• Protein concentration: For initial binding studies, use a concentration range of 1-100 nM for both endoglin and the TGF-β family ligands, based on published binding affinities.

• Co-factors: Include 1-2 mM calcium and/or magnesium ions as these divalent cations often enhance binding of TGF-β family members to their receptors.

• Detection methods: Consider using surface plasmon resonance (SPR), ELISA, or microscale thermophoresis (MST) for quantitative binding measurements.

• Controls: Include a negative control using heat-denatured Endoglin Human, Sf9 or a different protein from the same expression system to confirm binding specificity.

• Validation: Confirm the biological activity of Endoglin Human, Sf9 by measuring its ability to bind with rhTGF-beta RII/Fc in a functional ELISA, as this is a standard quality control method .

How can researchers troubleshoot and optimize Western blot detection of Endoglin Human, Sf9?

To optimize Western blot detection of Endoglin Human, Sf9:

• Sample preparation: Prepare samples in reducing conditions using buffer containing 5% β-mercaptoethanol or DTT, as endoglin is a disulfide-linked homodimer. Heat samples at 95°C for 5 minutes before loading.

• Gel percentage: Use 8-10% acrylamide gels to optimally separate the 90 kDa glycosylated protein.

• Transfer conditions: Implement wet transfer at 30V overnight at 4°C for large proteins to ensure complete transfer.

• Blocking: Use 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature to minimize background.

• Primary antibody: If using anti-His tag antibodies, ensure they recognize C-terminal His tags. For Endoglin-specific antibodies, select those recognizing the extracellular domain (Glu26-Gly586).

• Common issues and solutions:

  • Multiple bands: May represent different glycosylation states or partial degradation; reduce sample handling time and add protease inhibitors

  • No signal: Verify transfer efficiency with Ponceau S staining; if transfer is successful, increase antibody concentration or incubation time

  • High background: Increase washing steps and duration; reduce antibody concentration

• Controls: Include a positive control of commercial endoglin and a molecular weight marker to verify the 90 kDa band position .

What quality control methods should be employed to verify the structure and function of Endoglin Human, Sf9 before experimental use?

A comprehensive quality control approach for Endoglin Human, Sf9 should include:

• Purity assessment: Analyze by RP-HPLC and SDS-PAGE to ensure >95% purity as standard practice indicates. Coomassie blue or silver staining can be used depending on protein concentration .

• Identity confirmation: Perform Western blot analysis using specific anti-endoglin antibodies and anti-His tag antibodies to verify protein identity.

• Structural integrity: Employ circular dichroism (CD) spectroscopy to assess secondary structure elements, ensuring proper protein folding.

• Glycosylation analysis: Use PNGase F digestion followed by gel shift analysis to confirm glycosylation status and heterogeneity.

• Oligomeric state verification: Conduct native PAGE or size exclusion chromatography to confirm the homodimeric state of the protein.

• Functional testing: Verify biological activity through binding assays with known ligands:

  • TGF-β receptor binding using a functional ELISA

  • BMP-10 binding assays (the ED50 for this effect is 0.06-0.36 μg/mL in the presence of 100 ng/mL of Recombinant Human BMP-10)

• Endotoxin testing: Perform LAL assay to ensure endotoxin levels are sufficiently low for cell-based assays (<1 EU/μg protein) .

How should researchers interpret contradictory results when comparing endoglin's effects in different cell models?

When faced with contradictory results regarding endoglin's effects across different cell models:

• Context-dependent signaling: Recognize that endoglin's function may be cell type-specific due to varying expression levels of TGF-β receptors and downstream effectors. For example, endoglin's effects in endothelial cells (e.g., HUVECs) may differ from those in cancer cells (e.g., MDA-MB-231) due to different signaling network compositions .

• Experimental conditions analysis: Systematically compare experimental protocols, including:

  • Culture conditions (serum percentage, growth factors present)

  • Cell density and passage number

  • Duration of endoglin exposure

  • Concentration of recombinant endoglin used

• Cellular background consideration: Document the baseline expression of endoglin and TGF-β pathway components in each cell model using qPCR and Western blotting to identify potential compensatory mechanisms.

• Pathway crosstalk: Investigate potential interactions between TGF-β signaling and other pathways (like MAPK/p38) that may be differentially regulated across cell types, explaining divergent outcomes .

• Statistical validation: Perform meta-analysis across experiments using standardized effect sizes to determine if contradictions are statistically significant or within expected experimental variance.

• Resolution approach: Design experiments that directly compare cell models under identical conditions, potentially using co-culture systems to examine cell-cell interactions that may influence endoglin function.

What statistical approaches are most appropriate for analyzing data from angiogenesis assays using Endoglin Human, Sf9?

For angiogenesis assays involving Endoglin Human, Sf9:

• Tube formation quantification: When analyzing HUVEC tube formation parameters (total tube length, number of branches, loops formed):

  • Apply one-way ANOVA followed by post-hoc tests (Tukey or Bonferroni) for multiple group comparisons

  • Use paired t-tests when comparing just two conditions (e.g., with/without endoglin)

  • Consider non-parametric alternatives (Kruskal-Wallis or Mann-Whitney) if data does not follow normal distribution

• Concentration-response relationships:

  • Implement regression analysis to establish dose-dependent effects

  • Calculate EC50 values using non-linear regression models

• Time-course experiments:

  • Apply repeated measures ANOVA to account for temporal dependencies

  • Consider area under the curve (AUC) analyses to capture cumulative effects

• Multifactorial experimental designs:

  • Use two-way or three-way ANOVA when testing endoglin in combination with other factors (e.g., growth factors, inhibitors)

  • Employ post-hoc interaction analyses to identify synergistic or antagonistic effects

• Variability consideration:

  • Incorporate mixed-effects models when working with primary cells that may have donor-dependent variability

  • Report both biological and technical replicates separately

• Software selection:

  • Utilize specialized angiogenesis quantification software (e.g., WIMASIS Image Analysis) for standardized measurements

  • Export quantitative data for further statistical analysis in programs like SigmaPlot (version 12.3 or later) or R .

How can Endoglin Human, Sf9 be utilized in developing novel cancer therapeutics based on anti-angiogenic strategies?

Endoglin Human, Sf9 provides several avenues for developing anti-angiogenic cancer therapeutics:

• Therapeutic target validation: Use the recombinant protein in competition assays to evaluate the potential efficacy of anti-endoglin antibodies or small molecule inhibitors. Research shows that endoglin plays a major role in tumor neoangiogenesis, making it a promising target .

• Screening platform development: Establish high-throughput screening assays using Endoglin Human, Sf9 to identify compounds that disrupt its interaction with TGF-β family ligands or its co-receptors.

• Mechanistic studies: Employ the recombinant protein to elucidate endoglin's precise role in regulating endothelial cell proliferation, migration, and tube formation, providing insights for targeted intervention.

• Biomarker discovery: Utilize Endoglin Human, Sf9 to develop sensitive assays for detecting soluble endoglin in patient samples, potentially serving as a biomarker for monitoring anti-angiogenic therapy response.

• Combination therapy investigation: Test recombinant endoglin in combination with established anti-angiogenic drugs (e.g., VEGF inhibitors) to identify synergistic effects, as research indicates endoglin may modulate VEGF production through the TGF-β/SMAD3/VEGF signaling axis .

• Humanized antibody development: Build upon the existing humanized anti-endoglin antibodies that bind endoglin and inhibit vascular proliferation, which have demonstrated potential in treating conditions associated with angiogenesis, including cancer .

What emerging technologies could enhance our understanding of endoglin's role in tumor microenvironment and angiogenesis?

Several cutting-edge technologies can advance our understanding of endoglin in tumor angiogenesis:

• Single-cell RNA sequencing (scRNA-seq): Apply this technology to tumor samples to reveal heterogeneity in endoglin expression among different endothelial cell populations and identify novel cell-specific regulatory mechanisms.

• Spatial transcriptomics: Implement techniques like Visium or MERFISH to map endoglin expression patterns within tumor tissues while preserving spatial context, allowing correlation with hypoxic regions and vascular structures.

• CRISPR-based screening: Develop endothelial cell-specific CRISPR screens to identify genetic interactions with endoglin that modulate angiogenic responses, potentially revealing new therapeutic targets.

• Advanced in vivo imaging: Utilize intravital microscopy with fluorescently labeled Endoglin Human, Sf9 to track its distribution and dynamics in tumor vasculature in real-time.

• Organoid and microfluidic technologies: Create vascularized tumor organoids or tumor-on-a-chip platforms incorporating endoglin-manipulated endothelial cells to model tumor-vasculature interactions under controlled conditions.

• Artificial intelligence: Apply machine learning algorithms to image analysis of endoglin-stained tumor samples to identify patterns associated with tumor progression and treatment response.

• Zebrafish xenografts: Further develop zebrafish models for studying tumor angiogenesis, as these allow direct visualization of vascular sprouting in response to cancer cells with modified endoglin expression or exposure to Endoglin Human, Sf9 .

How might Endoglin Human, Sf9 contribute to understanding the role of endoglin in diseases beyond cancer?

Endoglin Human, Sf9 can be instrumental in investigating endoglin's role in various non-cancer conditions:

• Cardiovascular diseases: Use the recombinant protein to study endoglin's function in vascular remodeling and endothelial dysfunction, key processes in atherosclerosis and hypertension. Endoglin's known role in cardiovascular development suggests broader implications in adult cardiovascular pathologies .

• Fibrotic disorders: Investigate how endoglin modulates TGF-β signaling in fibroblasts and myofibroblasts, potentially contributing to fibrotic processes in organs like the liver, kidney, and lung.

• Ocular disorders: Explore endoglin's potential involvement in ocular neovascularization conditions, as humanized anti-endoglin antibodies have shown promise in treating macular degeneration, choroidal neovascularization, and proliferative vitreoretinopathy .

• Hereditary hemorrhagic telangiectasia (HHT): Utilize the recombinant protein to study how endoglin mutations lead to vascular malformations characteristic of HHT, potentially developing in vitro models of the disease.

• Diabetic complications: Examine endoglin's role in diabetic nephropathy and retinopathy, where aberrant angiogenesis contributes to disease progression. Humanized anti-endoglin antibodies have been investigated for treating diabetic nephropathy .

• Inflammatory disorders: Study how endoglin expression on activated macrophages might influence inflammatory responses and macrophage polarization in conditions like rheumatoid arthritis or inflammatory bowel disease.

• Preeclampsia: Investigate the role of soluble endoglin, which is elevated in preeclampsia, using the recombinant protein to model its effects on vascular function during pregnancy.

What are the recommended concentrations and experimental conditions for using Endoglin Human, Sf9 in different assay systems?

What are the key experimental controls that should be included when studying endoglin signaling using the recombinant protein?

When studying endoglin signaling with Endoglin Human, Sf9, include these essential controls:

• Vehicle control: Samples treated with the same buffer used for protein reconstitution (PBS with/without carrier protein) to account for buffer effects.

• Negative protein control: A similarly produced recombinant protein (preferably another ZP family protein) from the same expression system to control for Sf9-specific effects.

• Heat-denatured Endoglin Human, Sf9: To distinguish between specific biological activity and non-specific protein effects.

• Concentration gradient: Multiple concentrations of Endoglin Human, Sf9 to establish dose-dependency.

• Positive signaling control: Known TGF-β pathway activator (e.g., TGF-β1) to verify pathway responsiveness.

• Pathway inhibition control: Small molecule inhibitor of TGF-β receptor kinase activity (e.g., SB431542) to confirm signaling specificity.

• Genetic knockdown/knockout: siRNA against endoglin or endoglin-knockout cell lines to compare effects of exogenous versus endogenous protein.

• Time course controls: Samples collected at multiple timepoints to capture both immediate and delayed signaling events.

• Endoglin neutralization: Anti-endoglin antibody that blocks ligand binding, to confirm observed effects are due to the protein's binding activity .

• Carrier protein control: When using the non-carrier-free version, include equivalent amounts of the carrier protein (BSA) alone to account for potential carrier effects .