VSTM2L Antibody, FITC conjugated

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

VSTM2L is a protein that has been identified as a novel VDAC1 binding protein positively associated with prostate cancer progression. Recent research has established VSTM2L as a key regulator of ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation. VSTM2L forms a complex with VDAC1 (Voltage-Dependent Anion Channel 1) and HK2 (Hexokinase 2), preventing VDAC1 oligomerization and thereby inhibiting ferroptosis while maintaining mitochondrial homeostasis . This mechanism suggests that VSTM2L may serve as a potential therapeutic target for prostate cancer treatment.

What detection methods can be used with VSTM2L antibodies?

VSTM2L antibodies can be employed in various detection methods including:

• Immunofluorescence microscopy (particularly advantageous with FITC-conjugated antibodies)

• Western blotting for protein expression analysis

• Immunoprecipitation for protein-protein interaction studies

• Flow cytometry for quantitative single-cell analysis

• Immunohistochemistry for tissue sample analysis

Research has utilized these methods to demonstrate VSTM2L's interactions with key proteins like VDAC1 and HK2, as evidenced by co-immunoprecipitation assays that confirmed direct binding between VSTM2L and VDAC1 .

How should VSTM2L antibodies be stored and handled?

For optimal performance of FITC-conjugated VSTM2L antibodies:

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze-thaw cycles; aliquot upon first thaw if necessary

• Protect from prolonged light exposure during experimental procedures

• Follow manufacturer recommendations for reconstitution buffers

• Use within the recommended shelf life period to ensure consistent signal intensity

How can VSTM2L antibodies be applied to study its role in ferroptosis inhibition?

To investigate VSTM2L's role in ferroptosis regulation, researchers can employ FITC-conjugated VSTM2L antibodies in:

• Co-localization studies: Combining VSTM2L-FITC antibodies with mitochondrial markers and VDAC1 antibodies (using a different fluorophore) to visualize the spatial arrangement of these proteins during ferroptosis induction.

• Live-cell imaging: Monitoring VSTM2L redistribution in response to ferroptosis inducers like RSL3.

• Protein complex detection: Using proximity ligation assays with VSTM2L antibodies to quantify the formation of VSTM2L-VDAC1-HK2 complexes under various conditions.

• Flow cytometric analysis: Correlating VSTM2L expression levels with ferroptosis markers in cell populations.

Recent research demonstrated that VSTM2L knockdown in prostate cancer cells enhanced their sensitivity to RSL3-induced ferroptosis, suggesting antibody-based detection of VSTM2L could serve as a predictive biomarker for ferroptosis susceptibility .

What considerations should be made when using VSTM2L-FITC antibodies in multi-parameter flow cytometry?

When incorporating VSTM2L-FITC antibodies into multi-parameter flow cytometry panels, researchers should consider:

• Spectral overlap: FITC emission spectra overlap with PE and other fluorophores; proper compensation controls are essential.

• Fixation effects: Some fixation protocols may affect VSTM2L epitope recognition or FITC fluorescence intensity.

• Subcellular localization: As VSTM2L interacts with mitochondrial proteins, permeabilization protocols must be optimized to access intracellular compartments.

• Panel design: Include markers for mitochondrial function (e.g., TMRE) and ferroptosis (e.g., lipid peroxidation indicators) to correlate with VSTM2L expression.

• Controls: Include isotype controls conjugated to FITC to account for non-specific binding.

How can VSTM2L antibodies be utilized to study the VSTM2L-VDAC1-HK2 tripartite complex?

The interaction between VSTM2L, VDAC1, and HK2 represents a critical mechanism for ferroptosis regulation. To study this complex:

• Sequential immunoprecipitation: First precipitate with VSTM2L antibodies, then probe for VDAC1 and HK2, or vice versa.

• Proximity ligation assays: Visualize protein-protein interactions in situ using VSTM2L-FITC antibodies paired with antibodies against VDAC1 or HK2.

• FRET analysis: When paired with appropriate acceptor fluorophore-conjugated antibodies against VDAC1 or HK2, FITC-conjugated VSTM2L antibodies can be used for FRET to measure nanoscale proximity.

• Super-resolution microscopy: Techniques like STORM or PALM can be used with VSTM2L-FITC antibodies to visualize complex formation at the nanoscale.

Research has confirmed that VSTM2L forms a tripartite complex with VDAC1 and HK2, enhancing their binding affinity and preventing VDAC1 oligomerization .

What optimization steps are required for immunofluorescence staining with VSTM2L-FITC antibodies?

For optimal immunofluorescence results with VSTM2L-FITC antibodies:

• Fixation method comparison: Compare paraformaldehyde, methanol, and acetone fixation to determine which best preserves VSTM2L epitopes while maintaining cellular structure.

• Permeabilization optimization: Test different detergents (Triton X-100, saponin, digitonin) at various concentrations to balance access to intracellular VSTM2L without disrupting protein complexes.

• Antigen retrieval evaluation: Determine if heat-induced or enzymatic antigen retrieval improves staining, particularly for formalin-fixed tissues.

• Blocking protocol development: Optimize blocking solutions (serum type, BSA concentration) to minimize background fluorescence while preserving specific signal.

• Antibody titration: Perform serial dilutions to determine the optimal antibody concentration that maximizes signal-to-noise ratio.

How can researchers confirm VSTM2L antibody specificity in ferroptosis studies?

Verifying antibody specificity is crucial for confident interpretation of results:

• VSTM2L knockdown controls: Use siRNA or shRNA-mediated VSTM2L knockdown cells as negative controls; the research showed VSTM2L knockdown enhanced sensitivity to RSL3-induced ferroptosis .

• Overexpression validation: Compare staining in wild-type versus VSTM2L-overexpressing cells.

• Peptide competition assays: Pre-incubate the antibody with purified VSTM2L protein before staining to demonstrate specificity.

• Cross-reactivity testing: Confirm the antibody doesn't recognize related V-set domain-containing proteins.

• Multiple antibody comparison: Use antibodies targeting different VSTM2L epitopes to confirm staining patterns.

• Western blot correlation: Correlate immunofluorescence intensity with Western blot quantification across cell lines with varying VSTM2L expression.

What are the optimal parameters for detecting VSTM2L-FITC in flow cytometry experiments?

To achieve sensitive and specific detection of VSTM2L using FITC-conjugated antibodies in flow cytometry:

• Laser and filter configuration: Use 488 nm laser excitation with a 530/30 nm bandpass filter for optimal FITC detection.

• Voltage optimization: Determine the appropriate PMT voltage that places negative controls in the first decade of the logarithmic scale while maintaining resolution of positive populations.

• Cell concentration adjustment: Maintain 1-5 × 10^6 cells/mL to prevent coincident events while ensuring sufficient event rate.

• Dead cell discrimination: Include viability dyes compatible with FITC (e.g., far-red dead cell stains) to exclude false positive signals from autofluorescent dead cells.

• Acquisition rate calibration: Set flow rate to <1000 events/second to ensure accurate signal recording and minimize coincident events.

How can researchers address weak or inconsistent VSTM2L-FITC antibody signals?

When encountering suboptimal VSTM2L-FITC staining:

• Epitope masking assessment: Test if the VSTM2L-VDAC1-HK2 complex formation masks the antibody's target epitope, potentially requiring alternative fixation or detergent conditions.

• Signal amplification strategies: Consider tyramide signal amplification or secondary antibody approaches if direct FITC conjugation provides insufficient signal.

• Photobleaching prevention: Minimize light exposure during processing; use antifade mounting media and optimize image acquisition settings.

• Binding competition evaluation: Test if endogenous ligands compete with antibody binding, potentially requiring different lysis conditions or detergents.

• Fresh vs. frozen comparison: Compare staining efficiency between fresh and frozen samples to determine optimal sample preparation.

What controls are critical for validating VSTM2L antibody performance in ferroptosis research?

Essential controls include:

• VSTM2L knockout/knockdown cells: Critical negative controls that should show significantly reduced staining; research demonstrated VSTM2L knockdown enhanced RSL3-induced ferroptosis .

• Treatment-responsive cells: Cells treated with ferroptosis inducers like RSL3 should show changes in VSTM2L localization or complex formation patterns.

• Isotype-FITC controls: Essential for distinguishing specific binding from Fc receptor interactions or non-specific adherence.

• Antibody cross-reactivity panel: Test the antibody against related proteins, particularly other V-set domain-containing proteins.

• Cell line panel validation: Verify staining patterns across multiple cell lines with known VSTM2L expression profiles.

How should researchers analyze the relationship between VSTM2L expression and ferroptosis markers?

For comprehensive analysis:

• Correlation analysis: Calculate Pearson or Spearman correlation coefficients between VSTM2L-FITC signal intensity and markers of ferroptosis (lipid peroxidation, GSH depletion).

• Time-course experiments: Track VSTM2L expression, localization, and complex formation at multiple time points following ferroptosis induction.

• Multi-parameter gating strategy: In flow cytometry, develop gating strategies that correlate VSTM2L expression with mitochondrial integrity markers and cell death indicators.

• Intensity threshold determination: Establish signal intensity thresholds that distinguish true VSTM2L positivity from background.

• Single-cell analysis: Consider single-cell approaches to identify subpopulations with varying VSTM2L expression and ferroptosis susceptibility.

Research has shown that VSTM2L knockdown in prostate cancer cells reduced GSH levels, which was further decreased by RSL3 administration, suggesting a correlation between VSTM2L expression and ferroptosis resistance mechanisms .

How might VSTM2L antibodies contribute to developing ferroptosis-based cancer therapies?

VSTM2L antibodies could facilitate therapeutic development through:

• Patient stratification biomarker development: Determine if VSTM2L expression levels predict response to ferroptosis-inducing therapies in prostate cancer.

• Therapeutic antibody development: Investigate if antibodies targeting VSTM2L can disrupt its interaction with VDAC1 and HK2, potentially sensitizing cancer cells to ferroptosis.

• Drug screening assays: Develop high-throughput assays using VSTM2L antibodies to identify compounds that disrupt the VSTM2L-VDAC1-HK2 complex.

• Theranostic approaches: Explore VSTM2L antibody conjugates for combined imaging and therapeutic applications.

Research has established that VSTM2L suppression combined with RSL3 treatment significantly reduced tumor volume and weight in vivo, suggesting potential therapeutic applications .

What potential roles might VSTM2L have beyond ferroptosis regulation?

To explore additional VSTM2L functions:

• Interactome mapping: Use VSTM2L antibodies for immunoprecipitation followed by mass spectrometry to identify novel binding partners beyond VDAC1 and HK2.

• Subcellular localization studies: Employ fractionation and immunofluorescence to track VSTM2L distribution across cellular compartments under various conditions.

• Post-translational modification analysis: Investigate how phosphorylation, glycosylation, or other modifications affect VSTM2L function and complex formation.

• Tissue expression profiling: Characterize VSTM2L expression across normal and pathological tissues to identify potential roles in other diseases.