ANXA8L2 Antibody

What is ANXA8L2 and what cellular functions does it perform?

ANXA8L2 is a member of the annexin family of evolutionarily conserved Ca2+ and phospholipid binding proteins. The protein functions as an anticoagulant that indirectly inhibits the thromboplastin-specific complex . As with other annexin family members, ANXA8L2 likely plays roles in membrane organization, trafficking, and cellular signaling pathways dependent on calcium and phospholipid binding. Notably, overexpression of this gene has been associated with acute myelocytic leukemia, suggesting its potential role in cellular transformation . ANXA8L2 shares significant homology with ANXA8, which has been studied more extensively in cancer contexts .

What is the molecular weight of ANXA8L2 and how does it appear on Western blots?

ANXA8L2 has a calculated molecular weight of approximately 37 kDa (36,879 Da specifically according to some sources) . When detected by Western blot, ANXA8L2/ANXA8 typically appears as a specific band at approximately 36-37 kDa . For optimal detection, samples should be prepared under reducing conditions, and membranes are typically probed with the primary antibody at dilutions ranging from 1:500 to 1:2000 depending on the specific antibody used . The signal can be visualized using appropriate HRP-conjugated secondary antibodies specific to the host species of the primary antibody (e.g., anti-sheep IgG for AF8105 or anti-rabbit IgG for rabbit polyclonal antibodies) .

What applications are ANXA8L2 antibodies validated for?

ANXA8L2/ANXA8 antibodies have been validated for multiple research applications, with specific utility depending on the antibody clone. Common applications include:

• Western Blot (WB): Used to detect the presence and quantity of ANXA8L2 protein in cell or tissue lysates, typically at dilutions of 1:500-1:2000 .

• Immunohistochemistry (IHC): For localization of ANXA8L2 in tissue sections, with recommended dilutions of 1:50-1:200 .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ANXA8L2 in solution .

• Immunofluorescence (IF): For subcellular localization studies, typically used at dilutions of 1:50-1:100 .

Each application requires optimization of antibody concentration, incubation conditions, and detection methods for specific experimental contexts.

What tissues or cell lines can be used as positive controls for ANXA8L2 antibody validation?

Based on available data, several human cell lines and tissues serve as suitable positive controls for ANXA8L2/ANXA8 antibody validation:

• A549 human lung carcinoma cell line has been validated for detection of ANXA8 by Western blot .

• Human placenta tissue sections show specific ANXA8 expression, particularly in endothelial cells, making it a suitable control for IHC applications .

• Various renal cell carcinoma (RCC) samples show increased expression of ANXA8, suggesting their utility as positive controls .

When establishing new antibody validation protocols, researchers should consider including these known positive controls alongside appropriate negative controls to ensure specificity.

How can cross-reactivity between ANXA8L2 and other annexin family members be minimized in experimental design?

Cross-reactivity between closely related annexin family members presents a significant challenge in ANXA8L2 research. To minimize this issue:

• Select antibodies specifically validated against multiple annexin family members. For example, certain monoclonal antibodies like E9 and B7 have been specifically screened against human ANXA2 and ANXA5 to confirm ANXA8 specificity .

• Include appropriate controls in experimental designs:

  • Recombinant proteins of related annexin family members (ANXA2, ANXA5) as negative controls

  • Purified ANXA8L2/ANXA8 protein as positive control

  • ANXA8 knockout or knockdown samples when available

• When developing sandwich ELISA or other dual-antibody detection systems, use antibody pairs targeting different epitopes, as demonstrated with E9 and B7 monoclonal antibodies, which apparently target distinct regions of ANXA8 .

• For Western blot applications, optimize separation conditions to ensure adequate resolution between annexin family members of similar molecular weights.

What are the optimal expression systems for producing recombinant ANXA8L2 protein for antibody development or validation?

Research indicates two primary expression systems have been successfully employed for ANXA8/ANXA8L2 recombinant protein production:

• Pichia pastoris expression system:

  • Provides eukaryotic post-translational modifications

  • Has been successfully used for human ANXA8 expression for antibody screening and as an antigen

  • Particularly valuable for proteins requiring proper folding

• Escherichia coli expression systems:

  • E. coli BL21(DE3) strain has been used for murine ANXA8 expression

  • E. coli-derived recombinant human ANXA8/ANXA8 (Ala2-Pro327) has been utilized for antibody production

  • Typically provides higher yield but lacks eukaryotic post-translational modifications

For antibody development projects requiring highly specific antibodies, expressing the target protein in both systems provides complementary advantages – P. pastoris for properly folded protein most similar to native structure, and E. coli for higher yields needed for immunization protocols.

How might ANXA8L2 antibodies be optimized for detection of cancer biomarkers in clinical samples?

ANXA8/ANXA8L2 has shown potential as a tumor biomarker, with elevated expression observed in various cancer types including acute myelocytic leukemia and renal cell carcinoma . For optimizing antibodies in cancer biomarker applications:

• Sensitivity optimization:

  • Develop sandwich ELISA assays with paired antibodies targeting different epitopes

  • Current state-of-the-art ELISA systems can achieve detection limits as low as 0.065 ng/mL

  • Evaluate signal amplification strategies for detecting low abundance biomarkers in early-stage cancer

• Sample preparation considerations:

  • For tissue biopsies, optimize antigen retrieval protocols (e.g., heat-induced epitope retrieval using basic antigen retrieval reagents has shown success for ANXA8 detection in placenta samples)

  • For liquid biopsies, develop protocols to account for potential interference from plasma/serum components

• Validation across cancer subtypes:

  • Test antibody performance across multiple cancer types and stages to establish sensitivity and specificity profiles

  • Compare with established cancer biomarkers to determine added diagnostic or prognostic value

• Correlation with clinical outcomes:

  • Verify whether antibody-detected levels correlate with disease progression, as suggested by studies showing ANXA8 expression is associated with poor prognosis in renal cell carcinoma

What are the considerations for developing antibodies that can detect both human and mouse ANXA8L2 for translational research?

Developing cross-species reactive antibodies is valuable for translational research moving between mouse models and human applications. Key considerations include:

• Epitope selection strategy:

  • Target conserved regions between human and mouse ANXA8L2/ANXA8

  • Screen candidate antibodies against both human and mouse proteins

  • Research has successfully identified antibodies with cross-species reactivity to both human and mouse ANXA8

• Validation workflow:

  • Express both human and murine ANXA8L2 recombinant proteins

  • Use counter-screening approaches to select antibodies that specifically bind to ANXA8 from both species

  • Perform side-by-side Western blot, IHC, or ELISA validation with both human and mouse samples

• Application-specific optimization:

  • Different applications may require different antibody clones or concentrations for optimal cross-species detection

  • For IHC applications, tissue fixation and antigen retrieval protocols may need species-specific optimization

• Potential limitations:

  • Even with conserved epitopes, species differences in post-translational modifications may affect antibody binding

  • Quantitative comparisons between species should be approached with caution

What are the recommended protocols for using ANXA8L2 antibodies in Western blot applications?

Based on published research, optimized Western blot protocols for ANXA8L2/ANXA8 detection include:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration using BCA Protein Assay Reagent Kit

  • Load approximately 20-25 μg of protein per lane

• Electrophoresis conditions:

  • Separate proteins on 12% SDS-PAGE gels

  • Use reducing conditions for optimal results

• Transfer and blocking:

  • Transfer to PVDF membranes

  • Block in 5% non-fat dry milk in TBST at room temperature for 1 hour

• Antibody incubations:

  • Primary antibody: Incubate at dilutions ranging from 1:500-1:2000 (e.g., 2 μg/mL for AF8105) at 4°C overnight

  • Wash three times with TBST for 30 minutes

  • Secondary antibody: HRP-conjugated antibody matching the host species of primary antibody (e.g., anti-sheep for AF8105, anti-rabbit for rabbit polyclonal antibodies) at room temperature for 1 hour

• Detection:

  • Visualize using ECL detection systems

  • A specific band should be detected at approximately 36-37 kDa

What are the critical considerations for immunohistochemical detection of ANXA8L2 in tissue sections?

For optimal IHC detection of ANXA8L2/ANXA8 in tissue sections:

• Sample preparation:

  • Use paraffin-embedded tissue sections

  • For ANXA8 detection in human placenta, immersion fixation has shown good results

• Antigen retrieval:

  • Perform heat-induced epitope retrieval using basic antigen retrieval reagents

  • This step is critical as it significantly enhances antibody binding to the target epitope

• Antibody incubation:

  • Use 1 μg/mL antibody concentration (for AF8105) or dilutions of 1:50-1:200 for other antibodies

  • Incubate overnight at 4°C for optimal binding

• Detection system:

  • HRP-DAB detection systems have been validated for ANXA8 visualization

  • Counterstain with hematoxylin for tissue structure context

• Controls and interpretation:

  • Include positive controls where ANXA8 expression is expected (e.g., endothelial cells in placenta)

  • In placental tissue, ANXA8 shows specific localization to endothelial cells

  • For cancer studies, compare expression between tumor and adjacent normal tissue

How can sandwich ELISA systems be optimized for specific and sensitive detection of ANXA8L2?

Development of a sensitive and specific sandwich ELISA for ANXA8L2/ANXA8 requires:

• Antibody pair selection:

  • Use antibodies targeting different, non-overlapping epitopes

  • Specific monoclonal antibodies like E9 and B7 have demonstrated successful pairing for ANXA8 detection

  • Screen multiple antibody combinations to identify pairs with optimal sensitivity and specificity

• Recombinant protein standards:

  • Generate highly pure recombinant ANXA8L2 protein for standard curve development

  • Consider the expression system carefully as protein conformation may affect antibody binding

• Cross-reactivity elimination:

  • Validate against related annexin family members (ANXA2, ANXA5) to ensure specificity

  • Current optimized systems have achieved detection limits of approximately 0.065 ng/mL while maintaining specificity

• Sample preparation optimization:

  • Develop protocols for various sample types (cell lysates, tissue extracts, serum)

  • Identify and mitigate potential interfering substances in complex biological samples

• Assay validation:

  • Determine the linear range, lower limit of detection, precision (intra- and inter-assay variability)

  • Assess recovery in spiked samples and dilution linearity

What approaches can be used to study the functional implications of ANXA8L2 expression in cancer models?

To investigate ANXA8L2 functional roles in cancer:

• Expression modulation strategies:

  • Gene knockdown using siRNA or shRNA targeting ANXA8L2

  • This approach has been used in renal cell carcinoma studies to examine effects on cell cycle

  • CRISPR/Cas9-mediated knockout for complete elimination of expression

  • Overexpression systems using appropriate vectors for gain-of-function studies

• Phenotypic assays:

  • Cell proliferation assays to measure growth effects

  • Cell cycle analysis using flow cytometry (ANXA8 may influence cell cycle progression)

  • Migration and invasion assays to assess metastatic potential

  • Apoptosis assays to investigate cell survival roles

• Molecular interaction studies:

  • Co-immunoprecipitation using validated ANXA8L2 antibodies to identify protein binding partners

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Calcium-dependent binding studies to investigate phospholipid interactions

• In vivo models:

  • Xenograft models using cell lines with modulated ANXA8L2 expression

  • Patient-derived xenografts to maintain tumor heterogeneity

  • Correlation of ANXA8L2 expression with tumor growth, invasion, and response to therapy

• Pathway analysis:

  • Investigate downstream signaling effects through phosphoprotein arrays

  • Perform transcriptomic analysis to identify genes regulated by ANXA8L2 expression

  • Examine effects on calcium signaling pathways