EGFL7 Antibody, HRP conjugated

What is EGFL7 and what are its primary biological functions?

EGFL7 (Epidermal growth factor-like protein 7) is an endothelial cell-derived secreted factor that regulates vascular tube formation. It plays crucial roles in regulating vascular tubulogenesis in vivo, inhibiting platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, and promoting endothelial cell adhesion to the extracellular matrix and angiogenesis . While initially considered endothelial-specific, EGFL7 expression has been detected in tumor cells in various cancers, indicating pathological functions beyond normal vascular development .

What is EGFL7 Antibody, HRP conjugated and how does it differ from other EGFL7 antibodies?

EGFL7 Antibody, HRP conjugated is a rabbit polyclonal antibody against EGFL7 that has been conjugated with horseradish peroxidase (HRP). This conjugation allows for direct detection in immunoassays without requiring a secondary antibody . Unlike unconjugated antibodies that would require a secondary detection system, this HRP-conjugated version simplifies workflows by eliminating an additional incubation step, particularly beneficial in ELISA applications .

What are the common synonyms and identifiers for EGFL7?

EGFL7 is known by multiple synonyms in scientific literature:

• EGF-like protein 7

• Multiple epidermal growth factor-like domains protein 7 (MEGF7)

• NOTCH4-like protein

• Vascular endothelial statin (VE-statin)

• Zneu1

• UNQ187/PRO1449

Key identifiers include:

• UniProt ID: Q9UHF1 (human)

• HGNC: 20594

• OMIM: 608582

• KEGG: hsa:51162

What are the validated applications for EGFL7 Antibody, HRP conjugated?

The EGFL7 Antibody, HRP conjugated has been primarily validated for ELISA applications . While some polyclonal EGFL7 antibodies have broader application ranges including immunohistochemistry (IHC), the HRP-conjugated version is specifically optimized for enzyme-linked immunosorbent assays where direct detection is advantageous . For researchers requiring antibodies for multiple applications, verification of specific application compatibility is recommended before proceeding with experiments.

How should EGFL7 Antibody, HRP conjugated be stored and handled to maintain activity?

For optimal preservation of activity, EGFL7 Antibody, HRP conjugated should be stored at -20°C or -80°C immediately upon receipt . The antibody should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly degrade both the antibody and the HRP enzyme activity. The storage buffer typically contains 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When working with the antibody, maintain cold chain practices and return to appropriate storage promptly.

How can EGFL7 Antibody, HRP conjugated be used to investigate the relationship between hypoxia and EGFL7 expression?

Research has demonstrated that EGFL7 expression is oxygen-dependent and correlates with hypoxia-inducible factor 1-alpha (HIF-1α) levels . To investigate this relationship, researchers can design experiments where cells are cultured under normoxic and hypoxic conditions, followed by protein expression analysis using EGFL7 Antibody, HRP conjugated in ELISA. This approach can quantitatively measure EGFL7 upregulation in response to hypoxia. Parallel experiments measuring HIF-1α can establish correlation patterns. Studies have shown that among HIF-1α-positive hepatocellular carcinoma patients, 69% were medium positive and 31% were strong positive for EGFL7, indicating a mechanistic relationship between hypoxia and EGFL7 regulation .

What methodologies can be employed to study the interaction between EGFL7 and integrins using this antibody?

The EGFL7-ITGB3 (β3 integrin) axis has been identified as significant in multiple myeloma progression . To study this interaction, researchers can employ:

• Competitive binding assays using EGFL7 Antibody, HRP conjugated to detect displacement of EGFL7 binding by integrin inhibitors

• Pull-down assays followed by ELISA detection of protein complexes

• Functional assays measuring cell adhesion, migration, or proliferation in the presence of EGFL7 and integrin inhibitors (e.g., Cilengitide)

Research has shown that blockade of the EGFL7-ITGB3 axis can enhance therapeutic outcomes in multiple myeloma when combined with other treatments . Experimental designs should include appropriate controls such as siRNA knockdown of EGFL7 or ITGB3 to validate specificity.

What are common causes of high background when using EGFL7 Antibody, HRP conjugated in ELISA?

High background when using HRP-conjugated antibodies can result from several factors:

• Insufficient blocking: Optimize blocking buffer concentration (typically 5% non-fat dry milk in TBST has been effective with EGFL7 antibodies )

• Cross-reactivity: Validate specificity using appropriate negative controls (Jurkat cells have been identified as negative controls for EGFL7 expression )

• Excessive antibody concentration: Titrate antibody dilutions (1:1000 to 1:5000) to determine optimal signal-to-noise ratio

• Inadequate washing: Implement more stringent washing steps with appropriate buffer

• HRP stability issues: Ensure antibody hasn't undergone multiple freeze-thaw cycles

To troubleshoot, systematic modification of each parameter is recommended with appropriate positive and negative controls.

How can researchers validate the specificity of EGFL7 antibody detection in complex samples?

Validation of antibody specificity for EGFL7 detection can be achieved through multiple complementary approaches:

• Use of known positive samples (human placenta lysate and HUVEC whole cell lysate have been validated for EGFL7 expression)

• Inclusion of established negative controls (Jurkat cells are documented EGFL7-negative controls)

• Knockdown validation using EGFL7 siRNA in positive cell lines followed by antibody testing

• Competitive inhibition using recombinant EGFL7 protein

• Comparison with alternative EGFL7 antibody clones or detection methods

For Western blot applications, the expected molecular weight of EGFL7 should be consistent with literature reports (validated in PMID: 18497746) .

What considerations should be made when designing experiments to detect EGFL7 in serum samples?

When designing experiments to detect EGFL7 in serum samples, researchers should consider:

• Sample processing: Quick separation and storage of serum at -80°C is critical

• Dilution optimization: Serum samples may require specific dilutions (preliminary data shows linearity of dilution for EGFL7 in serum with recovery percentages of 90-102% at 1:1 dilution)

• Matrix effects: Serum components may interfere with antibody binding (average recovery percentage in serum samples is approximately 92%, range 85-98%)

• Reference ranges: Establish normal reference ranges from healthy controls

• Clinical correlation: EGFL7 serum levels have been significantly correlated with vascular invasion and extrahepatic metastasis in HCC patients (P<0.0001)

Commercial ELISA kits for human EGFL7 typically have detection ranges of 0.47-30 μg/mL with sensitivity around 0.12 μg/mL, which should be considered when designing experiments .

How is EGFL7 antibody research contributing to potential cancer therapies?

Research using EGFL7 antibodies has revealed potential therapeutic applications in cancer treatment:

• Humanized monoclonal antibodies against EGFL7 (clone h18F7) have been developed and tested in preclinical models

• Combination therapy of anti-EGFL7 with anti-VEGF treatments (Avastin) has shown enhanced inhibition of tumor growth compared to anti-VEGF treatment alone in xenograft models

• EGFL7 has been identified as a predictor for HCC survival and metastasis, positioning it as a promising biomarker and therapeutic target

• The EGFL7-ITGB3-KLF2 axis has been identified as enhancing survival of multiple myeloma cells, suggesting a targeted therapeutic approach

Current clinical trials are evaluating the effects of combined anti-EGFL7 and anti-VEGF therapy on tumor vascular function and growth, with the hypothesis that blocking EGFL7 signaling may provide additional angiogenesis inhibition and potentially normalize tumor vasculature .

What methodological approaches can be used to study EGFL7's role in endothelial-tumor cell interactions?

To investigate EGFL7's role in endothelial-tumor cell interactions, several methodological approaches can be employed:

• Co-culture systems: Endothelial cells (HUVECs) with EGFL7 knockdown or overexpression can be co-cultured with tumor cells (e.g., RPMI8226 myeloma cells) to assess interactions

• Conditioned media experiments: Media from EGFL7-expressing cells can be applied to tumor cells to assess paracrine effects

• In vivo models: Anti-EGFL7 antibody treatment in tumor-bearing mice can reveal systemic effects (e.g., humanized EGFL7 antibody at 1 mg/kg body weight injected every other day)

• Gene expression analysis: RT-PCR with primers specific for EGFL7 and related pathway components (e.g., ITGB3, KLF2) can elucidate molecular mechanisms

Experimental evidence shows that EGFL7 knockdown in HUVECs affects co-cultured myeloma cells, suggesting important paracrine signaling between endothelial and tumor cells .

What are the challenges in developing quantitative assays for EGFL7 detection in different tissue types?

Developing quantitative assays for EGFL7 across different tissue types presents several challenges:

• Expression heterogeneity: EGFL7 expression varies significantly between normal and tumor tissues, requiring wide dynamic range detection

• Cellular localization: Initially described as endothelial-specific, EGFL7 is now known to be expressed in tumor cells, complicating interpretation of positive signals

• Isoform variation: Potential isoforms or post-translational modifications may affect antibody recognition

• Tissue-specific matrix effects: Different tissue homogenates may contain varying levels of interfering substances

• Reference standards: Establishing appropriate positive controls for each tissue type (e.g., placenta for high expression, Jurkat cells for negative control)

Inter-assay precision testing has shown CV% values <10% for EGFL7 ELISA kits, while intra-assay precision typically shows CV% values <8%, indicating reliable quantification when properly optimized .