VSTM2L Antibody, Biotin conjugated

What is VSTM2L and why is it significant in research?

VSTM2L (V-set and transmembrane domain-containing protein 2-like protein, also known as C20orf102) is a protein that has gained significant attention in cancer research. Recent studies have shown that VSTM2L is upregulated in various cancers compared to matched normal tissues, making it a promising therapeutic target and biomarker . In colorectal cancer, VSTM2L overexpression has been associated with resistance to chemoradiotherapy (CRT) through promoting cell proliferation and inhibiting apoptosis . For cholangiocarcinoma, VSTM2L has emerged as a potential therapeutic target and soluble prognostic biomarker, where silencing VSTM2L expression has been shown to attenuate cancer cell viability and survival . Researchers studying these pathologies require specific antibodies, such as biotin-conjugated VSTM2L antibodies, to facilitate detection methods including ELISA and Western blot applications.

What applications are biotin-conjugated VSTM2L antibodies most suitable for?

Biotin-conjugated VSTM2L antibodies are particularly valuable for numerous applications where signal amplification is required. Based on the experimental methodologies in current research, these antibodies are most suitable for:

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection of VSTM2L in patient samples

• Western blot analysis for protein expression assessment in cell lines and tissue samples

• Immunohistochemistry (IHC) for detecting VSTM2L in formalin-fixed paraffin-embedded tissues

• Dot blot assays for rapid screening

• Immunoprecipitation studies to investigate protein-protein interactions

The biotin conjugation enables detection systems using streptavidin or avidin conjugates, providing enhanced sensitivity through signal amplification while maintaining lot-to-lot consistency in detection systems .

How should VSTM2L antibodies be validated before experimental use?

Proper validation of VSTM2L antibodies is critical for experimental reliability. Methodologically, researchers should employ a multi-step validation approach:

• Positive control verification: Test the antibody on samples with known VSTM2L expression, such as VSTM2L-transfected 293T cell lysates .

• Negative control comparison: Validate specificity by comparing with non-transfected cell lysates lacking VSTM2L expression .

• Band size confirmation: Verify that the detected protein matches the predicted molecular weight (approximately 22 kDa for VSTM2L) .

• Cross-reactivity assessment: Test against related proteins to ensure specificity.

• Application-specific validation: For each intended application (WB, ELISA, IHC), perform specific validation tests using appropriate controls.

Research findings indicate that validated VSTM2L antibodies should clearly distinguish between VSTM2L-transfected and non-transfected samples in Western blots, with a band appearing at approximately 22 kDa in VSTM2L-expressing samples .

What is the optimal protocol for using biotin-conjugated VSTM2L antibodies in ELISA?

For optimal ELISA performance with biotin-conjugated VSTM2L antibodies, researchers should follow this methodological approach based on published protocols:

• Plate coating: Coat 96-well plates with a primary capture antibody (typically a rabbit anti-VSTM2L polyclonal antibody) at a concentration of 1 μg/ml .

• Blocking: Block with an appropriate buffer containing 1-5% BSA to reduce non-specific binding.

• Sample addition: Add diluted samples (serum, plasma, or whole blood) and incubate.

• Detection antibody: Apply the biotin-conjugated VSTM2L antibody at an optimized dilution.

• Signal development: Add streptavidin-HRP conjugate followed by appropriate substrate.

• Measurement: Measure absorbance at 450 nm using a microplate reader .

Research has shown this approach effectively detects soluble VSTM2L in whole blood samples from cancer patients, with significantly higher levels observed in cholangiocarcinoma patients compared to healthy donors . This technique provides a quantitative assessment of VSTM2L levels that correlates with disease progression.

How can researchers troubleshoot non-specific binding when using biotin-conjugated antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. To methodologically address this issue:

• Optimize blocking conditions: Test different blocking agents (BSA, milk proteins, normal serum) at various concentrations (1-5%). For VSTM2L antibodies, a 10 mg/mL BSA solution has been effectively used in some protocols .

• Adjust antibody concentration: Titrate the biotin-conjugated VSTM2L antibody to find the optimal concentration that maximizes specific signal while minimizing background.

• Pre-absorption controls: Include controls where the antibody is pre-absorbed with recombinant VSTM2L protein to confirm specificity.

• Modify wash protocols: Increase wash stringency by adding low concentrations of detergents (0.05-0.1% Tween-20) to wash buffers.

• Buffer optimization: Test different antibody diluents, such as 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2 buffer systems as used in some commercial formulations .

Additionally, researchers should be aware of endogenous biotin in samples, which may interfere with detection. Using streptavidin blocking kits prior to adding biotin-conjugated antibodies can help mitigate this issue in biotin-rich samples.

What are the critical storage and handling conditions for maintaining biotin-conjugated VSTM2L antibody activity?

Proper storage and handling are essential for maintaining the activity of biotin-conjugated VSTM2L antibodies. Based on established protocols for similar reagents:

• Storage temperature: Store lyophilized antibodies at -20°C and reconstituted antibodies at 4°C for short-term use or aliquoted and stored at -20°C for long-term storage.

• Reconstitution protocol: Restore lyophilized antibodies with deionized water or equivalent buffer systems as recommended by manufacturers .

• Preservatives: Many commercial preparations contain 0.01% (w/v) sodium azide as a preservative , which helps maintain antibody stability.

• Aliquoting: Divide reconstituted antibodies into single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise biotin conjugation.

• Working dilution preparation: When preparing working dilutions, use freshly prepared buffers containing stabilizers such as BSA (typically at 1-5% concentration).

Published data indicate that properly stored biotin-conjugated antibodies maintain their activity with minimal loss of performance for at least 12 months when following these guidelines .

How can biotin-conjugated VSTM2L antibodies be utilized in patient-derived organoid models?

Patient-derived organoid models represent an advanced application of VSTM2L antibodies in cancer research. Methodologically, researchers can implement the following approach:

• Organoid establishment: Collect fresh tumor tissues from patients (e.g., colorectal cancer patients receiving preoperative chemoradiotherapy) and culture them as three-dimensional organoids .

• Treatment protocols: Expose organoids to relevant treatments (e.g., chemoradiotherapy) to model therapeutic responses.

• VSTM2L detection methodology:

  • Fix organoids with appropriate fixatives

  • Process for sectioning or prepare whole-mount specimens

  • Perform immunostaining with biotin-conjugated VSTM2L antibodies

  • Use streptavidin-conjugated fluorophores for visualization

• Parallel marker analysis: Simultaneously assess DNA damage markers like γ-H2AX to evaluate correlation with VSTM2L expression .

Research findings have demonstrated that patient-derived organoids with high VSTM2L expression showed reduced γ-H2AX expression when treated with chemoradiotherapy compared to organoids with low VSTM2L expression, suggesting a mechanism for therapy resistance . This application enables personalized therapeutic response prediction and mechanistic studies of VSTM2L function in a clinically relevant model system.

What methodological approaches can be used to study the relationship between VSTM2L expression and cancer therapy resistance?

To investigate VSTM2L's role in therapy resistance, researchers can employ several complementary methodological approaches:

• Gene expression manipulation:

  • Overexpression: Transfect cancer cells with VSTM2L-overexpressed lentivirus (LV-VSTM2L) alongside appropriate controls (LV-NC)

  • Silencing: Use siRNA targeting VSTM2L (si-VSTM2L) in cancer cell lines

• Functional assays:

  • Cell viability assessment using WST-1 assay following treatment with relevant therapeutic agents

  • Clone formation assays to evaluate survival capacity

  • DNA damage repair quantification through γ-H2AX immunostaining

  • Apoptosis assessment using flow cytometry or Western blot for apoptotic markers

• Signaling pathway analysis:

  • Western blot analysis of downstream effectors (e.g., IL-4 signaling pathway components)

  • RNA-sequencing to identify global expression changes

Research data has shown that overexpression of VSTM2L in colorectal cancer cells promotes resistance to chemoradiotherapy by enhancing cell proliferation and inhibiting apoptosis, while silencing VSTM2L expression in cholangiocarcinoma cells significantly reduces cell viability and survival . The molecular mechanism appears to involve downstream IL-4 signaling affecting cell proliferation and apoptosis processes in the context of colorectal cancer .

What considerations should be made when analyzing soluble VSTM2L as a biomarker in liquid biopsies?

Analyzing soluble VSTM2L in liquid biopsies requires careful methodological considerations:

• Sample collection and processing:

  • Standardize collection procedures for whole blood, serum, or plasma

  • Process samples consistently with defined centrifugation parameters

  • Store samples at appropriate temperatures (-80°C recommended for long-term storage)

• ELISA optimization for biotin-conjugated antibodies:

  • Coat plates with capture antibody (e.g., rabbit anti-VSTM2L polyclonal antibody at 1 μg/ml)

  • Detect bound VSTM2L using biotin-conjugated detection antibodies

  • Employ streptavidin-HRP conjugates for signal development

  • Include a standard curve using recombinant VSTM2L protein

• Data analysis and interpretation:

  • Compare patient samples with appropriate control groups (e.g., healthy donors)

  • Correlate VSTM2L levels with clinical parameters and outcomes

  • Consider potential confounding factors such as inflammatory conditions

Research has demonstrated that soluble VSTM2L is significantly elevated in whole blood samples from cholangiocarcinoma patients compared to healthy donors, suggesting its potential as a non-invasive biomarker .

How does VSTM2L expression correlate with clinical outcomes in colorectal cancer patients?

VSTM2L expression has significant correlations with clinical outcomes in colorectal cancer, particularly in patients receiving preoperative chemoradiotherapy (pCRT). Methodologically, this has been assessed through:

• Gene expression analysis: Analysis of GEO profiling datasets revealed significantly upregulated VSTM2L in non-responders to pCRT .

• Protein expression quantification: Immunohistochemistry analysis of tumor samples using VSTM2L antibodies demonstrates variable expression levels between responders and non-responders .

• Statistical correlation: Association analysis between VSTM2L expression and clinical parameters.

A comprehensive study of 186 rectal cancer patients showed the following correlations:

The data demonstrates that high expression of VSTM2L is significantly associated with poor tumor regression after pCRT (P = 0.030) and correlates with worse survival outcomes, suggesting VSTM2L could serve as a prognostic biomarker for rectal cancer patients receiving pCRT .

What methodologies can be used to investigate VSTM2L as a therapeutic target in cholangiocarcinoma?

Investigating VSTM2L as a therapeutic target in cholangiocarcinoma requires a multi-faceted methodological approach:

• Target identification and validation:

  • Iterative patient partitioning (IPP) calculation to identify potential therapeutic targets

  • Expression analysis across multiple cancer databases (HPA, CCLE, TCGA, GTEx, TIMER)

  • Comparative expression analysis between cholangiocarcinoma cells and normal biliary epithelial cells (HIBEpiC)

• Functional validation:

  • siRNA-mediated silencing of VSTM2L expression in cholangiocarcinoma cell lines

  • Assessment of cell viability using WST-1 assay following VSTM2L silencing

  • Analysis of intracellular signaling pathway alterations via Western blotting

  • Evaluation of key survival and apoptotic markers (e.g., Survivin, p53, Galectin-3)

• In silico analysis:

  • Pathway enrichment analysis to identify affected biological processes

  • Analysis of VSTM2L effect on positive regulation of cell growth

  • Liptak's z value calculation to evaluate the impact on patient prognosis

Research findings demonstrate that silencing VSTM2L expression significantly attenuates the viability and survival of cholangiocarcinoma cells by blocking intracellular signaling pathways, and in silico analysis confirms VSTM2L affects positive regulation of cell growth in cholangiocarcinoma . These methodologies collectively establish VSTM2L as a promising therapeutic target in cholangiocarcinoma.

What is the relationship between VSTM2L expression and DNA damage repair in the context of cancer therapy resistance?

The relationship between VSTM2L expression and DNA damage repair represents an important aspect of cancer therapy resistance. Methodologically, this can be investigated through:

• DNA damage marker assessment:

  • Quantification of γ-H2AX expression as a marker of DNA double-strand breaks

  • Comparison between high and low VSTM2L-expressing samples after treatment with DNA-damaging agents

• Patient-derived organoid models:

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

  • Treatment with DNA-damaging agents (radiation, chemotherapy)

  • Assessment of DNA damage response through immunostaining for γ-H2AX

• Mechanistic investigation:

  • Analysis of DNA repair pathway components (homologous recombination, non-homologous end joining)

  • Evaluation of cell cycle checkpoint activation in relation to VSTM2L expression

Research findings from rectal cancer patient-derived organoids demonstrate that high expression of VSTM2L significantly reduced γ-H2AX expression compared to organoids with low VSTM2L expression when treated with chemoradiotherapy . This suggests that VSTM2L may confer resistance to DNA-damaging therapies by enhancing DNA damage repair capacity or attenuating the DNA damage response. Further mechanistic studies would help elucidate the precise molecular pathways through which VSTM2L affects DNA damage repair processes in cancer cells.

How can researchers optimize Western blot protocols for VSTM2L detection using biotin-conjugated antibodies?

Optimizing Western blot protocols for VSTM2L detection using biotin-conjugated antibodies requires careful attention to several methodological parameters:

• Sample preparation:

  • Prepare protein lysates from cells or tissues using appropriate lysis buffers

  • Include protease inhibitors to prevent protein degradation

  • Determine optimal loading amount (typically 15-20 μL of cell lysate)

• Electrophoresis conditions:

  • Use appropriate percentage gels (12-15% recommended for VSTM2L's 22 kDa size)

  • Include positive controls (VSTM2L-transfected cell lysates) and negative controls (non-transfected cell lysates)

• Transfer and blocking:

  • Optimize transfer conditions for small proteins

  • Block with 5% BSA or non-fat milk in TBST

• Antibody incubation:

  • Apply biotin-conjugated anti-VSTM2L antibody at optimal concentration (typically 1 μg/mL)

  • Incubate at 4°C overnight for maximum sensitivity

  • Use streptavidin-HRP for detection

• Detection and analysis:

  • Use enhanced chemiluminescence for visualization

  • Confirm the predicted band size of 22 kDa for VSTM2L

  • Quantify expression levels using appropriate software

Published Western blot data shows clear detection of VSTM2L in transfected 293T cell lysates at the predicted molecular weight of 22 kDa, with no detectable band in non-transfected control lysates when using optimized protocols .

What methodological approaches can distinguish between membrane-bound and soluble forms of VSTM2L?

Distinguishing between membrane-bound and soluble forms of VSTM2L requires specialized methodological approaches:

• Subcellular fractionation:

  • Separate membrane, cytosolic, and nuclear fractions using differential centrifugation

  • Analyze VSTM2L distribution across fractions by Western blot

  • Use membrane and cytosolic markers (e.g., Na+/K+-ATPase, GAPDH) as controls

• Detection of soluble VSTM2L:

  • Process culture supernatants or biological fluids by centrifugation to remove cellular debris

  • Concentrate samples using ultrafiltration if necessary

  • Perform ELISA using capture antibody systems with biotin-conjugated detection antibodies

• Surface biotinylation assays:

  • Label cell surface proteins with biotin

  • Isolate biotinylated proteins using streptavidin pulldown

  • Analyze VSTM2L presence in biotinylated fraction to confirm membrane localization

• Immunofluorescence microscopy:

  • Perform dual staining with membrane markers and VSTM2L antibodies

  • Compare non-permeabilized and permeabilized conditions to distinguish surface from intracellular localization

Research has demonstrated that soluble VSTM2L can be detected in whole blood samples from cancer patients using ELISA techniques , while membrane-bound VSTM2L has been studied in cell culture models. The relationship between these two forms and their respective roles in cancer progression and therapy resistance represents an important area for future investigation.

How can multiplexed assays be developed using biotin-conjugated VSTM2L antibodies in combination with other cancer biomarkers?

Developing multiplexed assays using biotin-conjugated VSTM2L antibodies alongside other cancer biomarkers requires sophisticated methodological approaches:

• Multiplex immunohistochemistry (mIHC):

  • Use tyramide signal amplification (TSA) to enable multiple rounds of staining

  • Employ different fluorophore-conjugated streptavidin for detecting biotin-conjugated VSTM2L antibodies

  • Combine with antibodies against other markers (e.g., γ-H2AX, Ki-67, cleaved caspase-3)

  • Perform multispectral imaging and computational analysis

• Bead-based multiplex assays:

  • Couple capture antibodies to distinct bead populations

  • Use biotin-conjugated detection antibodies for VSTM2L and other targets

  • Employ differentially labeled streptavidin conjugates

  • Analyze using flow cytometry or specialized bead readers

• Multiplex ELISA platforms:

  • Optimize antibody pairs to minimize cross-reactivity

  • Develop spatial multiplexing in microwell formats

  • Employ sequential detection strategies with appropriate blocking steps between markers

• Tissue microarray analysis:

  • Prepare tissue microarrays with multiple patient samples

  • Perform parallel staining for VSTM2L and other markers

  • Correlate expression patterns with clinical outcomes

Research findings indicate that VSTM2L expression could be meaningfully analyzed alongside DNA damage markers like γ-H2AX , proliferation markers, and apoptosis indicators to provide comprehensive insights into cancer biology and treatment response. Such multiplexed approaches enable simultaneous assessment of multiple biological processes, enhancing the understanding of complex cancer mechanisms and potentially improving patient stratification for personalized medicine applications.