EFEMP2 Antibody

What is EFEMP2 and why is it an important research target?

EFEMP2, also known as Fibulin-4, is a 49 kDa (calculated) extracellular matrix glycoprotein that contains four EGF domains and six calcium-binding EGF domains. It plays critical roles in:

• Elastic fiber formation and connective tissue development

• Vascular patterning and collagen biosynthesis

• Determination of cell fate during development

EFEMP2 has emerged as a significant research target because it demonstrates context-dependent roles in cancer progression. It functions as a tumor suppressor in breast cancer , bladder cancer , and lung cancer , while potentially acting as an oncogene in other contexts like cervical cancer . Defects in EFEMP2 are also causative in autosomal recessive cutis laxa syndrome .

What is the expected molecular weight of EFEMP2 in Western blot experiments?

When conducting Western blot analysis for EFEMP2:

• Calculated molecular weight: 49 kDa (based on 443 amino acids)

• Observed molecular weight: Typically appears at 55-60 kDa

This discrepancy between calculated and observed molecular weights is primarily due to post-translational modifications, particularly glycosylation, as EFEMP2 is a glycoprotein . The glycosylation pattern may vary between different cell and tissue types, potentially causing slight variations in the observed molecular weight.

What are the optimal conditions for EFEMP2 antibody applications in different experimental techniques?

The following table summarizes recommended conditions for major applications:

For all applications, it is essential to optimize conditions for your specific experimental system, as sample type and endogenous EFEMP2 expression levels can significantly impact results .

How should EFEMP2 immunohistochemistry protocols be optimized for different tissue types?

When optimizing IHC protocols for EFEMP2 detection:

• Fixation and Processing:

  • Cell lines: 4% paraformaldehyde for 30 minutes

  • Tissues: Standard formalin fixation

  • Section thickness: 4-5 μm sections are typically optimal

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval is necessary

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with normal goat serum

  • Primary antibody incubation at 4°C overnight

  • Secondary antibody incubation for 30 minutes at room temperature

• Detection and Scoring:

  • DAB (3′,3-diaminobenzidine) for chromogenic detection

  • Counterstain with hematoxylin

  • Semi-quantitative scoring system:

    • Staining intensity: 0 (no stain) to 3 (strong stain)

    • Percentage of positive cells: 0 (0%) to 4 (76-100%)

    • Total score: 0-7 (low expression ≤3, high expression ≥4)

EFEMP2 typically shows cytoplasmic or stromal localization, appearing as brown granules when using DAB detection .

How does EFEMP2 expression vary across different cancer types, and what are the implications for research?

EFEMP2 shows remarkable context-dependent expression and function in different cancer types:

Research implications:

• Context-Specific Analysis:

  • Always include appropriate cancer-specific controls

  • Interpret results within the specific cancer context

• Secreted vs. Cellular Protein:

  • Analyze both cellular lysates and culture supernatants

  • EFEMP2 expression may be more pronounced in the secreted fraction

• Clinical Correlations:

  • Low EFEMP2 expression is an independent high-risk predictor of survival in multiple cancer types

  • In glioma, high EFEMP2 expression correlates with M0 macrophage assembly and poor prognosis

What methods can be used to investigate EFEMP2 post-translational modifications?

EFEMP2 undergoes several post-translational modifications that affect its function. Here are specialized techniques to detect these modifications:

• Glycosylation Analysis:

  • Lectin Blot: Use biotinylated lectins (e.g., VVL for O-GalNAc) followed by streptavidin-HRP detection

  • Enzymatic Deglycosylation: Treat samples with glycosidases and observe molecular weight shifts

  • Western Blot: Compare observed (55-60 kDa) vs. calculated (49 kDa) molecular weights

• Protein-Protein Interactions:

• Secretion Analysis:

  • Culture Media Analysis: Collect and concentrate culture supernatants

  • Western Blot Comparison: Compare cellular vs. secreted fractions

  • Research shows EFEMP2 may be more abundant in secreted fractions

These techniques provide valuable insights into EFEMP2's post-translational state and potential functional modifications in various disease contexts.

What are common issues in Western blot detection of EFEMP2 and how can they be resolved?

Optimization strategy:

• Start with validated positive controls (human heart tissue, mouse kidney tissue)

• Test a range of antibody dilutions (1:500, 1:1000, 1:2000)

• Compare different blocking reagents (BSA preferred for EFEMP2)

• For low signals, analyze both cell lysates and concentrated culture media

What essential controls should be included when validating a new EFEMP2 antibody?

A comprehensive validation approach should include:

• Expression Controls:

  • Positive tissue controls: Human heart tissue, mouse kidney tissue, human skin tissue

  • Positive cell controls: HT-29, K-562, JAR, SW480 cells

  • Knockdown system: Use EFEMP2-targeting shRNA (verified constructs available)

• Technical Controls:

  • Primary antibody controls: Dilution series, isotype control

  • Secondary antibody control: Omit primary antibody

  • Blocking peptide control: Pre-incubate with immunizing peptide

• Application-Specific Controls:

  • Western Blot: Molecular weight markers (expect 55-60 kDa), loading control

  • IHC: Both positive and negative tissue sections, antigen retrieval controls

  • IP: Input control, non-specific IgG control

• Method Validation:

  • Confirm findings across multiple applications (WB, IHC, IF)

  • Correlate protein detection with mRNA expression

  • Use multiple antibodies from different manufacturers when possible

Proper documentation of all validation parameters is essential for reproducibility in EFEMP2 research .

How can EFEMP2 antibodies be used to investigate signaling pathways in cancer research?

EFEMP2 interacts with several key signaling pathways that can be investigated using antibody-based techniques:

• Wnt/β-catenin Pathway in Bladder Cancer:

  • After EFEMP2 overexpression/knockdown, analyze:

    • β-catenin translocation (nuclear vs. cytoplasmic)

    • c-Myc and cyclin D1 expression changes

• EMT Markers in Lung Cancer:

  • EFEMP2 regulates epithelial-mesenchymal transition

  • Monitor following EFEMP2 manipulation:

    • E-cadherin, N-cadherin, Vimentin (EMT markers)

    • MMP2, MMP9 (invasion markers)

• AKT Signaling in Cervical Cancer:

  • EFEMP2 regulates cell proliferation through AKT pathway

  • Key markers:

    • p-AKT, total AKT, CYCLIN E1, p21/WAF1/Cip1

• Macrophage Assembly in Glioma:

  • EFEMP2 expression correlates with M0 macrophage assembly

  • Investigate immune microenvironment markers

  • Use bioinformatic approaches to analyze macrophage phenotypes

These approaches enable comprehensive investigation of EFEMP2's mechanistic role in diverse cancer contexts.

What emerging research directions involve EFEMP2 antibodies?

Recent advances suggest several promising research directions:

• EFEMP2 as a Prognostic Biomarker:

  • Low EFEMP2 expression correlates with poor prognosis in multiple cancers

  • High EFEMP2 expression in glioma correlates with worse survival

  • Development of standardized IHC scoring systems for clinical application

• Tumor Microenvironment Interactions:

  • EFEMP2's role in M0 macrophage assembly in glioma

  • Investigating EFEMP2-mediated extracellular matrix remodeling

  • Potential influence on immune cell recruitment and function

• Therapeutic Targeting:

  • Exploring the AKT pathway connection in cervical cancer

  • CDK4 inhibitors (e.g., Palbociclib) in EFEMP2-expressing cancers

  • Context-dependent therapeutic approaches based on EFEMP2's dual role

• EFEMP2 in Liquid Biopsies:

  • As a secreted protein, EFEMP2 may be detectable in serum

  • Development of ELISA-based detection methods

  • Potential as a minimally-invasive diagnostic marker

• Post-translational Modification Analysis:

  • Glycosylation patterns as functional regulators

  • GALNT14-dependent breast cancer cell invasion mediated by EFEMP2

  • Correlation between modification states and disease progression

These emerging directions highlight the importance of high-quality, well-validated EFEMP2 antibodies for advancing our understanding of this protein's complex roles in health and disease.