VSTM2L Antibody

What is VSTM2L and why is it significant in cancer research?

VSTM2L is a protein that has been identified as a key regulator in several cancer types. Recent studies have revealed VSTM2L's critical roles in:

• Prostate cancer progression: VSTM2L is positively associated with prostate cancer progression and acts as a key regulator of ferroptosis, an iron-dependent form of programmed cell death .

• Cholangiocarcinoma development: VSTM2L has been identified as a potential therapeutic target and prognostic soluble biomarker for cholangiocarcinoma .

• Chemoradiotherapy resistance: High expression of VSTM2L is linked to resistance to chemoradiotherapy in rectal cancer through downstream IL-4 signaling .

The protein's ability to protect cancer cells against ferroptosis by forming complexes with VDAC1 (Voltage-Dependent Anion Channel 1) and HK2 (Hexokinase 2) makes it particularly relevant for therapeutic development strategies .

What experimental applications are suitable for VSTM2L antibodies?

VSTM2L antibodies have been successfully employed in multiple experimental applications:

• Western Blotting (WB): Rabbit polyclonal antibodies have been validated for Western blot applications with human samples, typically used at concentrations of 1 μg/mL .

• Immunoprecipitation (IP): Both mouse monoclonal and rabbit polyclonal antibodies have been used in immunoprecipitation studies, particularly for investigating protein-protein interactions .

• Immunohistochemistry (IHC): Antibodies have been used at 1:500 dilution for formalin-fixed paraffin-embedded tissue sections .

• ELISA: Sandwich ELISA techniques using rabbit polyclonal antibodies as capture antibodies and mouse monoclonal antibodies as detection antibodies have been developed to measure soluble VSTM2L in whole blood samples .

How do I validate VSTM2L antibody specificity for my research?

To ensure antibody specificity, consider implementing these validation methods:

• Overexpression control: Use VSTM2L-transfected cell lysates (e.g., VSTM2L-transfected 293T cells) alongside non-transfected controls to confirm band specificity at the predicted molecular weight (22 kDa) .

• Knockdown verification: Compare antibody signal in cells with and without VSTM2L knockdown using siRNA or shRNA approaches. The signal should significantly decrease in knockdown samples .

• Cross-reactivity assessment: Test the antibody against related proteins to ensure it specifically detects VSTM2L rather than other members of the protein family.

• Multiple antibody comparison: Use antibodies from different sources or those targeting different epitopes of VSTM2L to validate consistent results .

How can VSTM2L antibodies be utilized to study ferroptosis mechanisms in cancer research?

VSTM2L antibodies have proven valuable for investigating ferroptosis resistance mechanisms in cancer cells:

Experimental approach for studying VSTM2L in ferroptosis:

• Protein expression correlation with ferroptosis markers:

  • Use Western blot to examine the correlation between VSTM2L expression and key ferroptosis markers like GPX4, whose protein levels decrease upon VSTM2L knockdown in prostate cancer cells .

  • Compare GSH levels between VSTM2L knockdown and control cells to assess ferroptosis induction .

• Subcellular localization studies:

  • Perform immunofluorescence staining to examine VSTM2L localization in mitochondria, as VSTM2L is primarily localized to mitochondria and interacts with VDAC1 .

  • Co-staining with mitochondrial markers can help visualize VSTM2L's role in maintaining mitochondrial morphology.

• Protein-protein interaction analysis:

  • Use co-immunoprecipitation with VSTM2L antibodies to pull down protein complexes containing VDAC1 and HK2 .

  • Apply immunofluorescence to visualize the dissociation of HK2 from VDAC1 after VSTM2L knockdown.

• Rescue experiments:

  • Combine ferroptosis inducers (e.g., RSL3) with VSTM2L knockdown to assess enhanced sensitivity to ferroptosis.

  • Test if ferroptosis inhibitors like ferrostatin-1 (Fer-1) can rescue the cell death caused by VSTM2L knockdown .

What methodologies are recommended for investigating VSTM2L as a biomarker in cancer patients?

Research on VSTM2L as a biomarker requires specific methodological considerations:

Protocol framework for biomarker studies:

• Sample collection and processing:

  • Collect whole blood samples from cancer patients and healthy donors following appropriate ethical guidelines.

  • Process samples consistently to avoid introducing variables that might affect VSTM2L detection .

• ELISA development for soluble VSTM2L:

  • Coat 96-well plates with rabbit anti-VSTM2L polyclonal antibody (1 μg/ml).

  • Use mouse anti-VSTM2L monoclonal antibody as the detection antibody.

  • Measure absorbance at 450 nm using a microplate reader .

• Correlation with clinical parameters:

  • Compare VSTM2L levels with tumor regression after treatment (e.g., preoperative chemoradiotherapy).

  • Perform Kaplan-Meier progression-free survival (PFS) and disease-free survival (DFS) analyses to correlate VSTM2L expression with patient outcomes .

• Validation in patient-derived models:

  • Establish patient-derived organoids to evaluate the association between VSTM2L expression and tumor response to treatments like chemoradiotherapy .

  • Use immunohistochemistry to quantify VSTM2L levels in patient samples and correlate with clinical outcomes.

What techniques are most effective for studying VSTM2L's role in treatment resistance?

VSTM2L has been implicated in chemoradiotherapy resistance, particularly in rectal cancer:

Experimental design for treatment resistance studies:

• Patient sample analysis:

  • Compare VSTM2L expression levels between responders and non-responders to treatment using quantitative real-time PCR and Western blotting.

  • Perform immunohistochemistry on biopsy tissues before treatment to evaluate predictive potential .

• Patient-derived organoid models:

  • Culture patient-derived organoids with varying VSTM2L expression levels.

  • Treat organoids with chemoradiotherapy and assess DNA damage markers such as γ-H2AX.

  • High VSTM2L expression significantly reduces γ-H2AX expression, indicating resistance to treatment .

• Overexpression studies:

  • Create stable VSTM2L-overexpressing cell lines using lentiviral vectors.

  • Compare cell proliferation, viability, clone formation, DNA damage repair, and apoptosis ability between VSTM2L-overexpressing and control cells after treatment .

• Pathway analysis:

  • Use RNA sequencing and Gene Set Enrichment Analysis (GSEA) to identify downstream mechanisms of VSTM2L overexpression.

  • In rectal cancer, VSTM2L induces resistance through downstream IL-4 signaling .

How can VSTM2L antibodies be optimized for detecting protein-protein interactions?

VSTM2L forms important complexes with VDAC1 and HK2, influencing ferroptosis resistance:

Protocol optimization for protein interaction studies:

• Co-immunoprecipitation (Co-IP) strategy:

  • Perform semi-endogenous co-IP using VSTM2L antibodies to pull down the tripartite complex of VSTM2L, VDAC1, and HK2.

  • For reverse confirmation, use HK2-specific antibodies to capture VDAC1 protein and assess how VSTM2L knockdown affects this interaction .

• Crosslinking approaches:

  • Apply protein crosslinkers before immunoprecipitation to stabilize transient interactions.

  • Optimize crosslinker concentration and reaction time to preserve physiologically relevant interactions.

• Proximity ligation assay:

  • Use paired antibodies (rabbit anti-VSTM2L and mouse anti-VDAC1 or mouse anti-HK2) to visualize protein-protein interactions in situ.

  • This technique can reveal spatial information about where in the cell these interactions occur.

• Immunofluorescence co-localization:

  • Perform immunofluorescence staining to visualize the co-localization of VSTM2L, VDAC1, and HK2.

  • Knockdown of VSTM2L has been shown to cause dissociation of HK2 from VDAC1 in prostate cancer cells .

What considerations are important when using VSTM2L antibodies for studying mitochondrial function?

Since VSTM2L plays a crucial role in mitochondrial homeostasis:

Mitochondrial study protocol recommendations:

• Subcellular fractionation:

  • Isolate mitochondrial fractions from cells to enrich for VSTM2L and its interacting partners.

  • Confirm purity using mitochondrial markers before performing immunoblotting with VSTM2L antibodies.

• Quantification of mitochondrial changes:

  • Use living mitochondria imaging to analyze parameters like mitochondria number, area, perimeter, form factor, and aspect ratio in cells with varying VSTM2L levels.

  • VSTM2L inhibition causes reduction in all these parameters .

• Ultrastructural examination:

  • Perform transmission electron microscopy to observe mitochondrial morphology changes.

  • VSTM2L knockdown causes mitochondrial shrinkage, increased membrane density, and diminished cristae - all characteristics of ferroptosis .

• Mitochondrial ROS assessment:

  • Measure mitochondrial reactive oxygen species (mtROS) levels, as VDAC1 oligomerization influences mtROS balance.

  • VSTM2L has been shown to maintain this balance by preventing VDAC1 oligomerization .

What are the optimal conditions for using VSTM2L antibodies in Western blotting?

For optimal Western blot results with VSTM2L antibodies:

• Sample preparation:

  • Use VSTM2L-transfected cell lysates as positive controls.

  • Include non-transfected cell lysates as negative controls.

  • Load approximately 15 μL of lysate per lane for optimal detection .

• Antibody dilution:

  • Use rabbit polyclonal VSTM2L antibody at a concentration of 1 μg/mL for Western blotting.

  • Optimize secondary antibody dilution to minimize background while maintaining signal strength .

• Band detection:

  • The predicted band size for VSTM2L is approximately 22 kDa.

  • Be aware of potential post-translational modifications that might affect migration patterns .

• Validation controls:

  • Include VSTM2L knockdown samples to confirm antibody specificity.

  • Consider using multiple antibodies targeting different epitopes to verify consistent results .

How should VSTM2L antibodies be used in xenograft tumor models?

For in vivo studies involving VSTM2L:

• Tumor model development:

  • Establish xenograft models using VSTM2L knockdown cancer cells compared to control cells.

  • Monitor tumor growth over time to assess the impact of VSTM2L on tumor progression.

• Tissue processing and staining:

  • Perform hematoxylin and eosin (H&E) staining to evaluate histomorphological changes.

  • Use immunohistochemical staining to assess protein levels of VSTM2L, proliferation markers (Ki67), and ferroptosis-related proteins (GPX4) .

• Combination therapy assessment:

  • Combine VSTM2L knockdown with ferroptosis inducers like RSL3 for enhanced anti-tumor effects.

  • VSTM2L knockdown tumors show reduced expression of Ki67 and GPX4, effects that are further enhanced by RSL3 treatment .

• Quantification approaches:

  • Quantify staining intensity using appropriate software.

  • Compare protein expression patterns between different treatment groups and correlate with tumor response.

What are the recommended protocols for detecting soluble VSTM2L in patient samples?

For biomarker development studies:

• ELISA setup for soluble VSTM2L:

  • Coat 96-well plates with rabbit anti-VSTM2L polyclonal antibody (1 μg/ml).

  • Block plates effectively to minimize non-specific binding.

  • Use mouse anti-VSTM2L monoclonal antibody as the detection antibody.

  • Develop with appropriate substrate and measure absorbance at 450 nm .

• Sample considerations:

  • Whole blood samples have been successfully used to detect soluble VSTM2L.

  • Establish appropriate sample storage and handling procedures to maintain protein integrity.

• Quality control measures:

  • Include standard curves using recombinant VSTM2L protein.

  • Run samples in duplicate or triplicate to ensure reproducibility.

  • Include appropriate positive and negative controls with each assay run.

• Data analysis:

  • Compare VSTM2L levels between patient groups (e.g., cancer patients vs. healthy donors).

  • Correlate findings with clinical parameters and outcomes for biomarker validation .

How can researchers address common issues when working with VSTM2L antibodies?

Common challenges and solutions:

What methods are recommended for quantifying VSTM2L expression in relation to cancer progression?

For comprehensive expression analysis:

• Multi-platform approach:

  • Integrate RNA-seq data with protein expression data for a comprehensive view.

  • Compare VSTM2L expression across different cancer stages and grades.

• Digital pathology tools:

  • Use digital pathology software to quantify VSTM2L immunohistochemical staining intensity.

  • Correlate with clinical features such as tumor regression after treatment.

• Survival analysis:

  • Perform Kaplan-Meier analysis to correlate VSTM2L expression with patient outcomes.

  • High VSTM2L expression has been associated with poor prognosis in both prostate and rectal cancer patients .

• Statistical validation:

  • Use appropriate statistical tests to validate the significance of findings.

  • For example, Liptak's z value has been used to show that VSTM2L expression worsens prognosis in cholangiocarcinoma patients .

How should conflicting data on VSTM2L's role in different cancer types be interpreted?

When analyzing VSTM2L across different cancers:

• Context-specific functions:

  • VSTM2L may have tissue-specific roles depending on the cancer type.

  • In prostate cancer, it primarily acts through ferroptosis resistance mechanisms .

  • In rectal cancer, it appears to function through IL-4 signaling pathways .

• Pathway analysis approach:

  • Conduct pathway analysis in each cancer type to identify common and divergent mechanisms.

  • Integrate findings across studies to build a comprehensive model of VSTM2L function.

• Technical considerations:

  • Evaluate whether methodological differences might explain conflicting results.

  • Consider differences in antibodies, detection methods, and experimental models used.

• Validation strategies:

  • Confirm findings using multiple experimental approaches.

  • Consider patient-derived models like organoids to bridge in vitro and clinical observations .

What emerging applications of VSTM2L antibodies show promise for cancer research?

Promising research frontiers:

• Therapeutic antibody development:

  • Development of antibodies that could neutralize VSTM2L function, potentially sensitizing cancer cells to ferroptosis-inducing therapies.

  • Design of antibody-drug conjugates targeting VSTM2L-expressing cancer cells.

• Liquid biopsy approaches:

  • Further development of sensitive ELISA techniques for detecting soluble VSTM2L in blood as a non-invasive biomarker.

  • Integration with other biomarkers to improve cancer detection and monitoring.

• Spatial proteomics:

  • Use of advanced imaging techniques combined with VSTM2L antibodies to map protein interactions within cellular compartments.

  • Investigation of how VSTM2L localization changes during cancer progression and treatment.

• Combination therapy strategies:

  • Use of VSTM2L antibodies to identify patients who might benefit from combined ferroptosis inducers and conventional therapies.

  • Development of personalized treatment approaches based on VSTM2L expression profiles.

What are the key methodological gaps in current VSTM2L research?

Areas needing further development:

• Standardized detection protocols:

  • Development of standardized, validated protocols for VSTM2L detection across different sample types.

  • Establishment of reference ranges for soluble VSTM2L in healthy individuals and various cancer types.

• Functional antibodies:

  • Development of antibodies that can functionally inhibit VSTM2L's interaction with VDAC1 and HK2.

  • Investigation of whether these antibodies can sensitize cancer cells to ferroptosis.

• Single-cell approaches:

  • Application of single-cell techniques to understand heterogeneity in VSTM2L expression within tumors.

  • Correlation of expression patterns with treatment response at the cellular level.

• In vivo imaging:

  • Development of labeled VSTM2L antibodies for in vivo imaging to monitor treatment response non-invasively.

  • Integration with other molecular imaging approaches for comprehensive tumor characterization.