AGBL2 Antibody

What is AGBL2 and what cellular functions does it have?

AGBL2 (ATP/GTP binding protein-like 2) is a carboxypeptidase involved in the tubulin tyrosination cycle, which regulates microtubule dynamics. The protein has been identified as a latexin-interacting protein with significant implications in cancer biology . AGBL2 plays a crucial role in regulating cellular processes related to cancer progression, particularly in cancer stem cells. Recent studies demonstrate that AGBL2 functions in pathways related to epithelial-to-mesenchymal transition (EMT) and may contribute to chemotherapy resistance mechanisms .

What types of AGBL2 antibodies are currently available for research?

AGBL2 antibodies are available with various specifications to meet different research needs:

Most commercially available AGBL2 antibodies are rabbit-derived polyclonal antibodies that recognize human AGBL2, with some cross-reacting with mouse protein . Antibodies are typically available unconjugated, though conjugated versions (with fluorophores like FITC or Alexa Fluor) can be sourced for specialized applications .

What are the validated applications for AGBL2 antibodies?

AGBL2 antibodies have been validated for multiple experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of AGBL2 in solution

• Western Blot (WB): For protein expression analysis in cell/tissue lysates

• Immunohistochemistry (IHC): For detection in formalin-fixed, paraffin-embedded tissues

• Immunofluorescence (IF): For subcellular localization studies

• Immunocytochemistry (ICC): For detection in cultured cells

Studies have successfully employed these applications to correlate AGBL2 expression with clinical outcomes in cancer patients and to investigate AGBL2's role in cancer stem cells .

What are the recommended protocols for AGBL2 immunohistochemistry?

For optimal AGBL2 detection in tissue samples:

• Prepare 4-μm-thick sections from formalin-fixed, paraffin-embedded tissues

• Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Block endogenous peroxidase with 3% hydrogen peroxide containing 1% normal horse serum (10 minutes, room temperature)

• Incubate with anti-AGBL2 antibody at 1:100 dilution (overnight, 4°C in moist chamber)

• Apply horseradish peroxidase-conjugated anti-rabbit secondary antibody (30 minutes)

• Develop color using AEC substrate

• Counterstain with Mayer's hematoxylin

• Use normal fetal and adult kidney samples as positive controls

This protocol has been validated in multiple studies examining AGBL2 expression in cancer tissues .

How should AGBL2 expression be evaluated in immunohistochemistry?

AGBL2 expression can be evaluated using the following semi-quantitative scoring system:

• Mild/0: <1% of neoplastic cells express AGBL2

• Intermediate/1+: ≥1% and <10% of neoplastic cells express AGBL2

• Strong/2+: ≥10% of neoplastic cells express AGBL2

Samples scoring intermediate (1+) or strong (2+) should be considered positive. For accurate assessment, it's recommended to examine multiple fields and score based on both intensity and distribution of staining . In some studies, AGBL2 expression was observed primarily in the cytoplasm of positive cells, and the pattern of expression (rather than just intensity) may have biological significance .

How does AGBL2 expression correlate with cancer progression and prognosis?

AGBL2 expression has significant correlations with multiple clinical parameters:

In multivariate analysis using the Cox regression test, AGBL2 has been identified as an independent prognostic factor in certain cancers . The co-expression of AGBL2 with other markers, such as RARRES1, can have synergistic effects on prognosis prediction, with some co-expression patterns indicating 11-15 times higher risk of cancer relapse .

What is the relationship between AGBL2 and cancer stem cells?

AGBL2 demonstrates heightened expression in CD44+/CD24- cancer stem cells (CSCs), particularly those that have undergone epithelial-to-mesenchymal transition (EMT) . Research indicates that:

• CSCs with high AGBL2 expression show enhanced tumor-initiating capacity in NOD/SCID mice

• AGBL2 expression is upregulated during EMT induction in CSCs

• AGBL2 expression correlates with chemotherapy resistance in CSCs

• AGBL2 may contribute to the maintenance of stemness properties in cancer cells

These findings suggest that AGBL2 could be a potential therapeutic target for eliminating cancer stem cells and preventing cancer recurrence and metastasis.

What experimental approaches can be used to study AGBL2 function in cancer cells?

Several validated methodologies have been employed to investigate AGBL2 function:

• siRNA Knockdown: Transfection with AGBL2-specific siRNA followed by functional assays to assess effects on proliferation, migration, and chemoresistance

• Gene Expression Analysis: Affymetrix array analysis to correlate AGBL2 expression with clinical outcomes (e.g., analyzing tumors from patients with poor prognosis vs. good prognosis)

• Co-immunoprecipitation: To identify protein-protein interactions, such as the interaction between AGBL2 and latexin

• Flow Cytometry: For isolation of CD44+/CD24- tumor cells to study AGBL2 expression in cancer stem cells

• Western Blot: For protein expression analysis, using anti-AGBL2 antibody at 1:500 dilution with appropriate controls

How does AGBL2 interact with latexin, and what are the implications for cancer therapy?

AGBL2 forms immune complexes with latexin, as demonstrated through multiple experimental approaches:

• Tandem affinity purification (TAP) of latexin complexes reveals AGBL2 as an interacting partner

• Immunoprecipitation of endogenous latexin co-precipitates AGBL2

• Reverse immunoprecipitation with AGBL2 antibodies pulls down latexin

This interaction has significant therapeutic implications:

• Latexin appears to inhibit AGBL2 function in the tubulin tyrosination cycle

• Overexpression of latexin may counteract AGBL2-mediated processes in cancer progression

• The AGBL2-latexin interaction represents a potential target for developing novel cancer therapeutics

Understanding this interaction pathway could lead to new strategies for targeting cancer stem cells that are resistant to conventional chemotherapy.

How can AGBL2 and co-expressed markers be used for patient stratification and treatment decisions?

Combined expression analysis of AGBL2 with other markers enables more precise patient stratification:

These expression patterns can guide clinical decision-making:

• High-risk patients may benefit from more aggressive surveillance for early detection of metastasis

• High-risk patients might be candidates for adjuvant therapy

• The expression profile could help identify patients who would benefit from targeted therapies

What are the technical challenges in working with AGBL2 antibodies in multi-parameter studies?

Researchers face several challenges when using AGBL2 antibodies in complex studies:

• Antibody Specificity: Ensuring antibodies specifically detect AGBL2 without cross-reactivity, especially in tissues with complex protein mixtures

• Epitope Accessibility: Different fixation methods may affect epitope exposure, requiring optimized antigen retrieval protocols

• Variability Between Antibody Lots: Batch-to-batch variation can affect consistency in long-term studies

• Multiplex Staining Compatibility: When co-staining with other markers (e.g., RARRES1), antibody compatibility must be carefully validated

• Scoring Standardization: Establishing consistent scoring criteria across different studies and pathologists

To address these challenges, rigorous controls (positive, negative, and isotype) should be included in all experiments, and antibodies should be validated using multiple detection methods.

How can AGBL2 expression analysis be integrated into cancer biomarker panels?

Integration of AGBL2 into biomarker panels requires consideration of several factors:

• Complementary Markers: AGBL2 shows enhanced prognostic value when combined with markers like RARRES1, suggesting optimal biomarker panels should include complementary proteins

• Tissue Microarray Approach: Using TMAs with multiple samples per tumor (2-5 biopsies from areas of different morphology/grade) improves representation of tumor heterogeneity

• Standardized Scoring: Implementing consistent scoring systems across laboratories enables reliable meta-analysis of results

• Clinical-Pathological Integration: Correlating AGBL2 expression with traditional parameters (stage, grade) provides more comprehensive risk assessment

• Statistical Validation: Employing multivariate analysis techniques like Cox proportional hazard regression model validates the independent prognostic value of AGBL2

The goal should be developing clinically applicable tests that can guide personalized treatment decisions based on a patient's unique tumor biology profile.