vat1 Antibody

What is VAT1 protein and what cellular functions does it perform?

VAT1 (vesicle amine transport protein 1 homolog) is a 41.9 kDa integral membrane protein that plays important roles in cellular transport and metabolism. It primarily localizes in the cytoplasm but also resides in the outer membrane of mitochondria . The protein functions in regulating the storage and secretion of neurotransmitters in nerve cell terminals . VAT1 has been increasingly recognized for its role in cancer biology, where it has been identified as a potential oncogene in various tumor types, particularly in glioblastoma where its overexpression is associated with tumor migration and poor prognosis . Recent research has revealed its involvement in immunological processes, where VAT1-related genes are functionally enriched in immune response pathways .

What are the optimal applications for VAT1 antibodies in experimental research?

VAT1 antibodies can be effectively utilized in multiple experimental applications with varying dilution requirements:

For IHC applications, antigen retrieval is best performed with TE buffer pH 9.0, though citrate buffer pH 6.0 can serve as an alternative . It's essential to titrate the antibody concentration in each testing system to obtain optimal results, as sample-dependent variations are common .

What species reactivity has been validated for commercially available VAT1 antibodies?

Commercial VAT1 antibodies have demonstrated cross-reactivity with multiple species, offering versatility for comparative studies:

When working with less common species, preliminary validation experiments are strongly recommended to ensure antibody performance in your specific experimental system .

What is the recommended storage protocol for maintaining VAT1 antibody functionality?

For optimal preservation of VAT1 antibody activity, store the antibody at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibody remains stable for one year after shipment. Aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory protocols . For antibodies in the 20μl size format, be aware that the solution contains 0.1% BSA as a stabilizer . When working with the antibody, avoid repeated freeze-thaw cycles to prevent protein denaturation and loss of binding affinity.

How does VAT1 expression correlate with immune cell populations in glioma research models?

Gene set variation analysis (GSVA) has revealed significant negative correlations between VAT1 expression and multiple immune cell populations in glioma models. This relationship has been consistently observed across multiple databases including CGGA and TCGA :

Interestingly, immunohistochemistry analysis of tumor tissues revealed that T cell infiltration was positively correlated with VAT1 expression, suggesting complex regulatory mechanisms in the tumor microenvironment . This apparent contradiction between database analysis and tissue staining highlights the importance of using multiple methodological approaches when investigating VAT1's role in tumor immunity.

What are the biological processes most significantly associated with VAT1 expression in cancer models?

Gene ontology (GO) analyses from both CGGA and TCGA databases have identified distinct biological processes associated with VAT1 expression levels . These processes demonstrate a clear dichotomy between immune/inflammatory functions and normal neurophysiological processes:

These findings indicate that VAT1 overexpression may contribute to the immunosuppressive tumor microenvironment in gliomas while simultaneously downregulating normal neuronal functions . This makes VAT1 a potential target for immunotherapeutic approaches in glioma treatment. When designing experiments to investigate these pathways, researchers should consider both immune and neurological readouts to capture the full spectrum of VAT1's biological effects.

What methodological considerations are critical when using VAT1 antibodies for cancer immunotherapy research?

When investigating VAT1 as a potential immunotherapy target, several methodological considerations are essential:

• Expression analysis correlation: Integrate transcriptomic and proteomic approaches to confirm VAT1 expression patterns. Gene expression should be validated at the protein level using well-characterized antibodies .

• Immune checkpoint correlation: Recent studies have identified that immune checkpoint expression increases with VAT1 expression, suggesting VAT1 may function as part of the immune evasion machinery in tumors . Design experiments that simultaneously evaluate VAT1 and immune checkpoint molecules.

• Antibody validation for specific tumor types: For glioma research, VAT1 antibodies should be validated in IDH wild-type samples, where VAT1 is highly enriched according to both CGGA and TCGA databases .

• Combined in vitro and in vivo approaches: Supplement cell line studies with tissue microarray analysis to evaluate VAT1 expression in relation to immune cell infiltration patterns.

• Patient stratification considerations: High VAT1 expression is associated with poor prognosis in glioblastoma patients . When designing translational studies, stratify patient samples based on VAT1 expression levels to identify potential responders to immunotherapy.

What are the recommended experimental controls when using VAT1 antibodies in immunohistochemistry?

For reliable IHC results with VAT1 antibodies, implement these essential controls:

• Positive tissue controls: Include mouse pancreas tissue, which has been validated for VAT1 antibody reactivity in IHC applications . Brain tissue from multiple species (human, mouse, rat, pig) can also serve as positive controls .

• Antigen retrieval optimization: Compare results between TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine optimal conditions for your specific tissue samples .

• Antibody dilution series: Test a range of dilutions (1:500-1:2000) to establish the optimal signal-to-noise ratio for your experimental system .

• Isotype controls: Include mouse IgG1 isotype controls at equivalent concentrations to rule out non-specific binding .

• VAT1 knockout/knockdown validation: When possible, include tissue from VAT1 knockdown models to confirm antibody specificity.

• Cross-reactivity assessment: If studying multiple species, validate antibody performance in each species separately rather than assuming cross-reactivity.

How can VAT1 antibodies be employed in studying the tumor microenvironment and immunosuppression?

VAT1 has emerged as a significant factor in tumor-mediated immunosuppression, particularly in gliomas. To effectively investigate this relationship, researchers can employ the following methodological approaches:

• Multiparameter flow cytometry: Use VAT1 antibodies in combination with immune cell markers to quantify correlations between VAT1 expression and immune cell populations within the tumor microenvironment .

• Spatial transcriptomics integration: Combine VAT1 IHC with spatial transcriptomics to map the relationship between VAT1 expression zones and immune cell localization within tumors.

• Co-culture systems: Develop in vitro co-culture systems with VAT1-expressing tumor cells and immune cells to assess direct immunomodulatory effects.

• Checkpoint inhibitor combination studies: Investigate whether VAT1 blockade can enhance the efficacy of established checkpoint inhibitors, given the correlation between VAT1 and immune checkpoint expression .

• Single-cell analysis: Apply single-cell RNA sequencing to identify specific cell populations within tumors that express VAT1 and determine their immunological characteristics.

This research direction is particularly promising given that VAT1-related genes are functionally involved in immune responses, and differential expression analysis shows strong enrichment in immunological processes .

What are the technical challenges in developing antibodies against different VAT1 epitopes for research applications?

Developing antibodies against specific VAT1 epitopes presents several technical challenges that researchers should consider:

• Protein conformation considerations: VAT1's localization in both cytoplasm and mitochondrial outer membrane means antibodies must be designed to recognize epitopes accessible in different cellular compartments .

• Cross-reactivity management: Given VAT1's conservation across species, designing species-specific antibodies requires careful epitope selection to balance cross-reactivity with specificity .

• Isoform recognition: Consider whether your research requires antibodies that discriminate between potential VAT1 isoforms or post-translational modifications.

• Functional domain targeting: For functional studies, developing antibodies against specific functional domains may provide more mechanistic insights than general anti-VAT1 antibodies.

• Validation across applications: Antibodies that perform well in Western blot may not necessarily work in IHC or flow cytometry due to differences in protein conformation and fixation effects .

The current monoclonal antibodies against VAT1 fusion protein (such as Ag29812) demonstrate excellent specificity but researchers pursuing specialized applications may need to develop custom antibodies targeting specific epitopes relevant to their research questions .

What are the common troubleshooting strategies for inconsistent VAT1 antibody staining in immunohistochemistry?

When encountering variable results in VAT1 IHC applications, consider these methodological optimizations:

• Antigen retrieval modification: If standard TE buffer (pH 9.0) yields inconsistent results, alternate with citrate buffer (pH 6.0) to determine optimal retrieval conditions for your specific tissue samples .

• Dilution optimization: The recommended dilution range for IHC is broad (1:500-1:2000). Conduct a systematic titration series to determine the optimal concentration for your specific tissue type and fixation protocol .

• Sample preparation standardization: Variations in fixation time, fixative composition, and tissue processing can significantly impact antibody performance. Standardize these parameters across experimental samples.

• Detection system enhancement: For tissues with low VAT1 expression, consider amplification systems such as tyramide signal amplification or polymer-based detection methods.

• Background reduction strategies: If high background is observed, implement additional blocking steps using bovine serum albumin or normal serum from the secondary antibody host species.

• Tissue-specific protocol adjustment: VAT1 expression and accessibility may vary between tissue types. For example, brain tissue may require different protocols than pancreatic tissue, which is a validated positive control .

How can researchers optimize Western blot protocols for detecting VAT1 in different experimental systems?

For optimal Western blot detection of VAT1 across various experimental systems, implement these protocol refinements:

• Dilution range exploration: VAT1 antibodies show a remarkably wide effective dilution range (1:5000-1:50000), suggesting high sensitivity . Begin optimization at mid-range (1:25000) and adjust based on signal intensity.

• Sample preparation considerations:

  • For brain tissue: Homogenize in RIPA buffer with protease inhibitors, centrifuge at 14,000g, and collect the supernatant

  • For cultured cells (HSC-T6, HepG2, PC-12, NIH/3T3): Direct lysis in 2X Laemmli buffer typically yields good results

• Molecular weight expectations: VAT1 appears at 42-50 kDa on Western blots, with slight variation between species and cell types . Design gels that provide good resolution in this range.

• Loading control selection: For VAT1 analysis, GAPDH (37 kDa) may run too close to VAT1. Consider β-actin (42 kDa) with careful resolution or larger proteins like α-tubulin (55 kDa) as loading controls.

• Transfer optimization: Due to VAT1's moderate size, standard PVDF membranes with 0.45 μm pore size are suitable, with transfer at 100V for 60-90 minutes in standard Towbin buffer.

• Detection system selection: For the wide dilution range of VAT1 antibodies, enhanced chemiluminescence (ECL) detection systems provide sufficient sensitivity while allowing economical use of primary antibody.

How can VAT1 antibodies contribute to understanding glioma immunobiology and potential therapeutic targets?

VAT1 antibodies have become valuable tools for investigating the immunosuppressive mechanisms in gliomas, with several key research applications:

• Prognostic biomarker validation: High VAT1 expression correlates with poorer survival outcomes in glioma patients . Researchers can use VAT1 antibodies to stratify patient samples and correlate expression levels with clinical outcomes and treatment responses.

• Immunosuppressive pathway elucidation: Gene ontology analysis reveals that VAT1-related genes are functionally involved in immune responses . VAT1 antibodies can help map these pathways through co-immunoprecipitation and immunoblotting to identify binding partners in the immunosuppressive cascade.

• Therapeutic target assessment: As VAT1 contributes to glioma-induced immunosuppression, antibodies can be used to evaluate VAT1 as a novel target for immunotherapy . This can include developing blocking antibodies or using existing antibodies to monitor treatment effects.

• IDH mutation correlation studies: VAT1 is highly enriched in IDH wild-type gliomas according to both CGGA and TCGA databases . VAT1 antibodies can be used in comparative studies between IDH-mutant and wild-type tumors to understand differential mechanisms of immune evasion.

• Immune cell infiltration analysis: Despite database analyses showing negative correlations between VAT1 expression and immune cells, tissue IHC revealed positive correlation between VAT1 and T cell infiltration . This contradiction can be explored using multiplex immunofluorescence with VAT1 antibodies to characterize the spatial relationship between VAT1-expressing cells and infiltrating immune populations.

What are the current limitations in using VAT1 antibodies for neurodegenerative disease research?

While VAT1's role in regulating neurotransmitter storage and secretion suggests potential relevance to neurodegenerative diseases, several limitations exist in current antibody applications:

• Limited validation in neurodegenerative models: Most VAT1 antibody validation has focused on cancer models, particularly gliomas . Researchers need to independently validate antibody performance in neurodegenerative disease tissues and models.

• Specificity challenges in protein aggregates: Neurodegenerative diseases often feature protein aggregates that can cause non-specific antibody binding. Additional controls are necessary when studying VAT1 in tissues with pathological protein accumulation.

• Post-translational modification detection: Potential disease-specific post-translational modifications of VAT1 in neurodegenerative conditions may not be detected by current antibodies, which are primarily raised against recombinant proteins or fusion constructs .

• Synaptic localization resolution: VAT1's role in neurotransmitter regulation requires high-resolution imaging of synaptic structures . Current antibodies may not be optimized for super-resolution microscopy applications necessary for detailed synaptic studies.

• Cross-reactivity with aggregate-associated proteins: Researchers must carefully validate VAT1 antibodies against potential cross-reactivity with amyloid-beta, tau, alpha-synuclein, or other aggregation-prone proteins common in neurodegenerative disease samples.

To address these limitations, researchers should perform rigorous antibody validation in their specific neurodegenerative models and consider developing specialized antibodies for detecting disease-specific VAT1 modifications or conformations.