MVK Antibody

What is MVK and what cellular functions does it perform?

MVK (Mevalonate kinase) is a 42 kDa cytoplasmic protein belonging to the GHMP kinase family . It catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which serves as a key intermediate in the biosynthesis of isoprenoids and sterols, including cholesterol . The human MVK protein consists of 396 amino acid residues with a calculated molecular weight of 42.5 kDa .

MVK plays crucial roles in:

• Cholesterol biosynthesis pathway

• Steroid hormone production

• Production of other essential biomolecules critical for cellular membrane integrity

• Isoprenoid biosynthesis

• Cellular signaling pathways

Defects in the MVK gene can lead to metabolic disorders such as mevalonic aciduria (MEVA) and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS) .

What are the common applications of MVK antibodies in research?

MVK antibodies are utilized in various research applications:

The optimal dilution is sample-dependent and may require titration for each experimental system to obtain optimal results .

What is the reactivity profile of commercially available MVK antibodies?

Based on the search results, MVK antibodies demonstrate various reactivity profiles:

Some antibodies show cross-reactivity with samples from multiple species, which can be advantageous for comparative studies between human and animal models .

How is MVK involved in disease pathology?

MVK plays significant roles in several disease contexts:

• Metabolic Disorders: Mutations in the MVK gene can cause mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS), characterized by the accumulation of mevalonic acid due to impaired conversion to 5-phosphomevalonic acid .

• Cancer Biology: MVK is upregulated in lung adenocarcinoma tissues compared to normal tissues, and its expression can be induced by constitutively activated Kras . Elevated MVK expression correlates with poor prognosis in lung adenocarcinoma patients but not in lung squamous cell carcinoma patients .

• Immune Regulation: MVK interacts with TBK1, inhibiting TBK1 phosphorylation and thereby suppressing cGAS-Sting signaling . This suggests a non-metabolic function of MVK in modifying the immunological milieu.

• Inflammatory Conditions: MVK gene polymorphisms have been investigated in relation to ankylosing spondylitis, suggesting potential involvement in autoinflammatory processes .

What are the best practices for optimizing Western blot protocols for MVK detection?

When optimizing Western blot protocols for MVK detection, consider the following methodological approaches:

• Sample Preparation:

  • Use cell lines known to express MVK such as HepG2, A431, PC-3, Caco-2, or Jurkat cells .

  • Include appropriate lysis buffers with protease inhibitors to prevent protein degradation.

• Antibody Selection and Dilution:

  • Start with recommended dilutions (typically 1:1000-1:4000 for WB) .

  • For initial validation, consider testing multiple antibodies targeting different epitopes of MVK.

• Running Conditions:

  • Use reducing conditions as most validation data for MVK antibodies are generated under reducing conditions .

  • MVK typically appears at approximately 42 kDa band .

• Controls:

  • Positive controls: Use cell lysates with confirmed MVK expression (HepG2, A431) .

  • Negative controls: Consider using samples with MVK knockdown or from knockout models .

• Detection Method:

  • Both chemiluminescence and fluorescence-based detection methods are suitable.

  • For multiplex detection, consider using MVK antibody conjugates or secondary antibodies with different fluorophores .

Studies have successfully detected MVK protein using these approaches, with validated results across multiple cell lines and tissue samples .

How can I design experiments to study MVK's role in the cGAS-Sting signaling pathway?

Based on recent research findings, MVK inhibits the cGAS-Sting signaling pathway through interaction with TBK1 . To investigate this relationship, consider the following experimental design:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation (Co-IP) experiments using antibodies against MVK and TBK1 to verify their interaction .

  • GST pull-down assays using GST-tagged MVK fusion proteins to identify binding domains .

  • Domain mapping experiments to determine which region of MVK interacts with TBK1 (research indicates the C-terminus of MVK interacts with TBK1) .

• Functional Analysis:

  • Overexpress MVK in relevant cell lines (e.g., A549, H23) and measure IFNα/IFNβ mRNA levels by qPCR .

  • Knockdown MVK expression using siRNA or CRISPR-Cas9 and assess changes in IFNα/IFNβ expression .

  • Evaluate TBK1 phosphorylation at Ser172 under conditions of MVK overexpression or knockdown following HT-DNA transfection .

• In Vivo Models:

  • Utilize MVK-knockdown tumor models in immunocompetent mice to assess CD8+ T cell infiltration using immunofluorescence staining .

  • Correlate MVK expression levels with T-cell infiltration in tumor tissues .

• Controls and Validation:

  • Include appropriate controls for each experiment (empty vector controls, scrambled siRNA, etc.).

  • Validate antibody specificity using knockout or knockdown samples.

  • Confirm findings using multiple cell lines or primary cells.

This experimental approach has demonstrated that MVK interacts with TBK1, inhibits TBK1 phosphorylation, and consequently suppresses the cGAS-Sting signaling pathway, affecting immune cell infiltration in tumors .

What are the recommended methods for validating MVK antibody specificity?

Validating MVK antibody specificity is crucial for ensuring reliable experimental results. Consider these methodological approaches:

• Genetic Approaches:

  • Use siRNA or shRNA to knockdown MVK expression and confirm reduced signal in Western blot or immunofluorescence .

  • Employ CRISPR-Cas9 to generate MVK knockout cells as negative controls.

  • Overexpress tagged MVK (e.g., Flag-tagged) and confirm co-localization or co-detection with the MVK antibody .

• Peptide Competition Assays:

  • Pre-incubate the antibody with the immunizing peptide or recombinant MVK protein.

  • A specific antibody will show reduced or absent signal when blocked with its cognate antigen.

• Cross-Validation with Multiple Antibodies:

  • Use multiple antibodies targeting different epitopes of MVK.

  • Consistent detection patterns across different antibodies increase confidence in specificity.

• Immunoprecipitation-Mass Spectrometry:

  • Perform immunoprecipitation with the MVK antibody followed by mass spectrometry.

  • Confirm MVK as the predominant protein identified in the precipitate.

• Cell and Tissue Panel Screening:

  • Test the antibody across multiple cell lines with known MVK expression profiles .

  • Verify expected expression patterns in relevant tissues.

• Heterologous Expression Systems:

  • Express MVK in cell lines with low endogenous expression.

  • Confirm increased signal that correlates with expression levels.

Published studies have successfully validated MVK antibodies using these approaches, particularly in lung adenocarcinoma and immune signaling research contexts .

How can I investigate the relationship between MVK expression and Kras activation in cancer models?

Recent research has identified a link between constitutively active Kras and MVK expression in lung adenocarcinoma . To investigate this relationship, consider the following experimental approach:

• Cell Line Selection:

  • Use cell lines harboring wild-type Kras (e.g., H1299, H1793) and those with constitutively active Kras mutations .

  • Compare MVK expression levels between these cell lines using validated MVK antibodies.

• Genetic Manipulation:

  • Overexpress constitutively active Kras (e.g., KrasG12C) in wild-type cell lines and assess changes in MVK expression using Western blot .

  • Perform MVK knockdown in Kras-mutant cells to determine if MVK is necessary for Kras-induced phenotypes.

• Functional Assays:

  • Soft agar assays to assess anchorage-independent growth .

  • Cell proliferation, migration, and invasion assays to evaluate malignant phenotypes.

  • Combine Kras overexpression with MVK knockdown to determine if MVK is required for Kras-induced transformation .

• Patient Sample Analysis:

  • Compare MVK expression in lung adenocarcinoma tissues between patients with wild-type Kras and those with constitutively active Kras mutations .

  • Use immunohistochemistry with validated MVK antibodies to assess expression patterns in tissue samples .

• Signaling Pathway Analysis:

  • Investigate the effect of Kras activation on MVK expression regulatory mechanisms.

  • Assess downstream effectors that might mediate the relationship between Kras and MVK.

This approach has successfully demonstrated that constitutively active Kras induces MVK expression, and MVK expression is essential for the functional effects of constitutively active Kras in promoting malignant phenotypes in lung adenocarcinoma .

What techniques can be used to study MVK's role in immune cell infiltration in tumor microenvironments?

MVK has been shown to negatively correlate with CD8+ T-cell infiltration in tumors . To investigate this relationship, consider these methodological approaches:

• Multiplex Immunofluorescence Staining:

  • Perform multiplex immunofluorescence staining on tumor sections using antibodies against MVK and immune cell markers (CD3, CD8) .

  • This technique allows for simultaneous detection of multiple proteins in the same tissue section.

  • Follow validated protocols for tissue preparation, including proper dewaxing, antigen retrieval, and blocking steps .

• In Vivo Models:

  • Generate MVK-knockdown cancer cells (e.g., using shRNA or CRISPR-Cas9).

  • Implant these cells in immunocompetent mice (e.g., C57 mice).

  • Compare tumor growth and immune cell infiltration between MVK-knockdown tumors and control tumors .

  • Harvest tumors and analyze immune cell infiltration using immunofluorescence or flow cytometry.

• Flow Cytometry Analysis:

  • Disaggregate tumor tissues to obtain single-cell suspensions.

  • Stain for MVK (intracellular) and various immune cell markers.

  • Quantify correlations between MVK expression and immune cell populations.

• Transcriptomic Analysis:

  • Perform RNA-seq on tumors with varying levels of MVK expression.

  • Analyze gene expression patterns related to immune cell markers and immune response pathways.

  • Validate findings at the protein level using MVK antibodies in immunohistochemistry or Western blot.

• Public Database Analysis:

  • Analyze public databases to assess correlations between MVK expression and immune cell infiltration markers across cancer types .

  • Validate findings in independent cohorts.

Research has demonstrated that knockdown of MVK in cancer cells leads to increased CD8+ T-cell infiltration in tumors, suggesting a role for MVK in modulating the tumor immune microenvironment .

How can I study the interplay between MVK and TBK1 in regulating immune responses?

The interaction between MVK and TBK1 has important implications for immune signaling through the cGAS-Sting pathway . To investigate this relationship:

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation using antibodies against MVK to pull down TBK1 (or vice versa) .

  • Use GST-MVK fusion proteins in pull-down assays to confirm direct interaction .

  • Map interaction domains using truncated constructs of MVK protein (research indicates the C-terminus of MVK interacts with TBK1) .

• Phosphorylation Analysis:

  • Assess TBK1 phosphorylation at Ser172 using phospho-specific antibodies.

  • Compare phosphorylation levels under conditions of MVK overexpression or knockdown.

  • Stimulate cells with HT-DNA to activate the cGAS-Sting pathway and measure the effect of MVK on TBK1 phosphorylation .

• Downstream Signaling Analysis:

  • Measure expression of IFNα and IFNβ mRNA using qRT-PCR in cells with MVK overexpression or knockdown .

  • Assess activation of IRF3 and other downstream effectors of the cGAS-Sting pathway.

  • Evaluate changes in immune response gene expression profiles.

• Structural Studies:

  • Use structural biology approaches to characterize the MVK-TBK1 interaction interface.

  • Consider computational modeling to predict interaction sites.

  • Validate predictions using site-directed mutagenesis of key residues.

• Functional Consequences:

  • Assess the impact of MVK-TBK1 interaction on antiviral responses.

  • Evaluate effects on cancer cell immune evasion mechanisms.

  • Investigate potential therapeutic implications of modulating this interaction.

Research has demonstrated that MVK interacts with TBK1 and inhibits its phosphorylation, thereby suppressing cGAS-Sting signaling and potentially contributing to immune evasion in cancer contexts .

How do I resolve inconsistent MVK detection in Western blot experiments?

Inconsistent detection of MVK in Western blot experiments can be addressed through these methodological approaches:

• Sample Preparation Optimization:

  • Ensure complete lysis with appropriate buffers (e.g., RIPA buffer with protease inhibitors).

  • Verify protein concentration using reliable methods (BCA or Bradford assay).

  • Use fresh samples or properly stored (-80°C) aliquots to prevent protein degradation.

• Antibody Selection and Handling:

  • Test multiple MVK antibodies from different sources targeting different epitopes .

  • Follow manufacturer's recommended storage conditions (typically -20°C) .

  • Aliquot antibodies to avoid freeze-thaw cycles.

  • Validate antibody specificity using positive control lysates (HepG2, A431, PC-3) .

• Protocol Optimization:

  • Test a range of antibody dilutions (e.g., 1:1000-1:4000 for Western blot) .

  • Optimize blocking conditions to reduce background (typically 5% non-fat milk or BSA).

  • Adjust incubation times and temperatures for primary and secondary antibodies.

  • Consider using gradient gels for better separation around the 42 kDa region.

• Signal Enhancement:

  • Use signal enhancement methods compatible with your detection system.

  • For HRP-conjugated antibodies, optimize ECL substrate exposure times.

  • Consider using fluorescent secondary antibodies for more quantitative analysis.

• Controls and Validation:

  • Include positive control lysates from cells known to express MVK (HepG2, Jurkat) .

  • Use MVK knockdown samples as negative controls .

  • Consider running gradient gels to better resolve proteins around the 42 kDa range.

If inconsistent results persist, consider consulting published protocols specific to MVK detection, such as those referenced in studies investigating MVK in cancer and immune signaling contexts .

What factors should I consider when comparing results from different MVK antibodies?

When comparing results from different MVK antibodies, consider these important factors:

• Epitope Differences:

  • Different antibodies may target distinct regions of the MVK protein.

  • Antibodies targeting different epitopes may show varied accessibility depending on protein conformation or interactions.

  • Check the immunogen information to understand which region of MVK each antibody targets .

• Antibody Format and Properties:

  • Compare monoclonal vs. polyclonal antibodies (monoclonals offer higher specificity but may be more sensitive to epitope masking) .

  • Consider host species differences (rabbit vs. mouse) which may affect background in certain samples .

  • Evaluate conjugated vs. unconjugated formats and their compatibility with your detection systems .

• Validation Data Interpretation:

  • Review the validation data provided by manufacturers for each antibody .

  • Check published literature citing each antibody to assess performance in contexts similar to your research.

  • Consider the specific cell lines or tissues used in validation studies and their relevance to your experimental system.

• Application-Specific Performance:

  • An antibody performing well in Western blot may not be optimal for immunofluorescence or immunohistochemistry.

  • Evaluate application-specific dilution recommendations (e.g., 1:1000-1:4000 for WB vs. 1:10-1:100 for IF) .

  • Consider fixation sensitivity for applications like IF/IHC.

• Standardized Comparison:

  • Test multiple antibodies under identical experimental conditions.

  • Use the same positive and negative controls for fair comparison.

  • Document and report differences in detection patterns to contribute to the research community's knowledge.

How is MVK research contributing to our understanding of cancer immunology?

Recent research has revealed important connections between MVK and cancer immunology:

• MVK as an Immune Regulator in Cancer:

  • MVK has been shown to interact with TBK1 and inhibit cGAS-Sting signaling, a key pathway in anti-tumor immunity .

  • Knockdown of MVK in cancer cells leads to increased CD8+ T-cell infiltration in tumors, suggesting MVK may contribute to immune evasion mechanisms .

  • Public database analyses have demonstrated a negative correlation between MVK expression and T-cell infiltration in lung cancer tissues .

• Connection to Oncogenic Signaling:

  • MVK expression is upregulated in lung adenocarcinoma tissues compared to normal tissues .

  • Constitutively activated Kras (a common oncogenic driver) induces MVK expression in lung cancer models .

  • MVK appears to be essential for the functional effects of constitutively active Kras in promoting malignant phenotypes .

• Methodological Approaches:

  • Researchers are utilizing MVK antibodies for immunofluorescence staining to assess correlations between MVK expression and immune cell infiltration in tumor tissues .

  • Genetic manipulation of MVK expression in cancer cells, followed by in vivo tumor implantation, allows assessment of effects on tumor growth and immune response .

  • Co-immunoprecipitation studies using MVK antibodies have revealed important protein-protein interactions with immune signaling components .

• Future Research Directions:

  • Investigating MVK as a potential therapeutic target to enhance anti-tumor immunity.

  • Exploring the relationship between MVK and response to immunotherapy treatments.

  • Developing biomarker strategies using MVK antibodies to predict immunotherapy response.

  • Elucidating the complete mechanistic understanding of how MVK regulates immune signaling beyond TBK1 interaction.

These findings suggest that MVK has important non-metabolic functions in modifying the immunological milieu and may represent a new target for cancer immunotherapy approaches .

What are the emerging applications of MVK antibodies in autoimmune and inflammatory disease research?

MVK has been implicated in several autoimmune and inflammatory conditions, leading to new applications for MVK antibodies in these research areas:

• Mevalonate Kinase Deficiency (MKD) Research:

  • MVK antibodies are being used to study protein expression and function in patients with mevalonic aciduria and hyperimmunoglobulinemia D syndrome (HIDS) .

  • These conditions are characterized by periodic fevers and inflammatory episodes due to MVK mutations.

  • Antibodies allow assessment of MVK protein levels in patient samples to correlate with disease severity and presentation.

• Ankylosing Spondylitis Studies:

  • MVK gene polymorphisms have been investigated in relation to ankylosing spondylitis (AS) .

  • MVK antibodies can help determine if these genetic variations affect protein expression or function.

  • Research has examined potential connections between MVK and autoinflammatory mechanisms in AS .

• Isoprenoid Pathway in Inflammation:

  • The mevalonate pathway, where MVK functions, has been implicated in various inflammatory processes.

  • MVK antibodies allow researchers to study how alterations in this pathway affect inflammatory responses.

  • This research direction may reveal new therapeutic targets for inflammatory conditions.

• Methodological Approaches:

  • Immunohistochemistry using MVK antibodies can reveal expression patterns in inflammatory tissues .

  • Western blot analysis of MVK in patient-derived cells can correlate protein levels with disease markers.

  • Combined genetic and protein analysis using sequencing and antibody-based detection can provide comprehensive understanding of MVK-related disorders.

• Future Research Directions:

  • Developing diagnostic applications of MVK antibodies for inflammatory disorders.

  • Investigating MVK as a biomarker for disease activity or treatment response.

  • Exploring connections between metabolic pathways and inflammatory signaling through MVK function.

These emerging applications demonstrate the expanding utility of MVK antibodies beyond basic research into clinically relevant investigations of autoimmune and inflammatory conditions .