PMVK Antibody

What is PMVK and why is it significant in research?

Phosphomevalonate kinase (PMVK) is a cytosolic enzyme that catalyzes the conversion of mevalonate 5-phosphate into mevalonate 5-diphosphate in the fifth reaction of the cholesterol biosynthetic pathway . PMVK is highly expressed in heart, liver, skeletal muscle, kidney, and pancreas, with slightly lower expression in brain, placenta, and lung . Its significance lies in its participation in isopentenyl diphosphate biosynthesis via the mevalonate pathway, which is critical for cholesterol synthesis, protein prenylation, and other essential cellular processes . Recent research has also implicated PMVK in β-catenin signaling, suggesting its importance extends beyond cholesterol biosynthesis to roles in cellular development and potentially carcinogenesis .

What types of PMVK antibodies are available for research applications?

PMVK antibodies are available in multiple formats:

Each antibody type offers specific advantages depending on the experimental requirements, with polyclonals providing broader epitope recognition and monoclonals offering higher specificity .

What are the recommended applications for PMVK antibodies?

PMVK antibodies have been validated for multiple applications with specific recommended dilutions:

The observed molecular weight of PMVK in most applications is approximately 22 kDa, consistent with its calculated molecular weight of 22 kDa from its 192 amino acid sequence .

What species reactivity should be considered when selecting a PMVK antibody?

When selecting a PMVK antibody, species reactivity is a critical consideration that depends on your experimental model:

Sequence conservation of PMVK across species should be considered when interpreting cross-reactivity claims, as even single amino acid differences may affect antibody performance .

How can PMVK antibodies be utilized to investigate β-catenin signaling pathways?

Recent research has revealed that PMVK plays a critical role in regulating β-catenin signaling. When investigating this pathway, researchers should consider:

• Co-immunoprecipitation experiments using PMVK antibodies to pull down protein complexes containing both PMVK and β-catenin components

• Sequential immunoblotting to detect PMVK and β-catenin pathway proteins

• Implementation of PMVK knockdown studies using shRNAs to evaluate downstream effects on β-catenin signaling

Research has shown that PMVK knockdown significantly reduces β-catenin signaling, suggesting it is required for normal embryonic development . When designing such experiments, researchers should include appropriate controls for antibody specificity, such as PMVK knockout or knockdown cells, to confirm signal specificity .

What considerations should be made when using PMVK antibodies in cancer research studies?

PMVK has emerging roles in cancer biology, particularly in hepatocellular carcinoma (HCC). When designing cancer research studies:

• Compare PMVK expression in tumor versus normal adjacent tissue using carefully validated antibody dilutions

• Combine PMVK immunostaining with markers of the mevalonate pathway and β-catenin signaling

• Consider parallel studies with PMVK inhibitors (e.g., PMVKi5) to correlate expression with functional outcomes

Research has identified a small molecular inhibitor of PMVK (PMVKi5) that reduces HCC growth in vivo, suggesting therapeutic potential . When interpreting PMVK immunostaining in cancer tissues, the metabolic state of the tumor should be considered, as the mevalonate pathway activity may vary with proliferation rate, hypoxia, and other tumor microenvironment factors .

How should researchers validate PMVK antibody specificity for their particular experimental system?

Rigorous validation of PMVK antibody specificity is essential for reliable results and should include:

• Western blot analysis showing a single band at the expected molecular weight (22 kDa)

• Positive controls using cell lines known to express PMVK (e.g., HeLa, HepG2, Huh7, Hep3B)

• Competitive blocking experiments with the immunizing peptide

• Ideally, comparison of results from multiple antibodies targeting different PMVK epitopes

• Genetic validation using PMVK knockdown (shRNA) or knockout models

For advanced validation, consider testing reactivity in cells expressing PMVK mutants, particularly those affecting key residues like Lys17, Ser20, Gly21, Lys22, Asp23, Arg73, and Arg141, which have been identified as functionally important .

What methodologies are recommended for investigating PMVK mutations in genetic disorders?

PMVK mutations have been linked to autosomal dominant disseminated superficial porokeratosis . When investigating such genetic connections:

• Combine genetic sequencing with protein expression analysis using validated PMVK antibodies

• Develop site-directed mutagenesis constructs expressing disease-associated PMVK variants

• Assess enzymatic activity in parallel with expression level changes

• Use immunohistochemistry with PMVK antibodies on patient samples to correlate genotype with protein expression patterns

The specificity of the antibody becomes particularly important when examining mutant forms of PMVK, as conformational changes might affect epitope accessibility. Western blotting under both reducing and non-reducing conditions may provide insights into structural alterations caused by mutations .

What are the optimal storage and handling conditions for maintaining PMVK antibody activity?

Proper storage and handling are critical for maintaining antibody performance:

For lyophilized antibodies, reconstitute according to manufacturer specifications (typically with 0.2ml distilled water to yield 500μg/ml) . Most PMVK antibodies are supplied in PBS with preservatives such as 0.02% sodium azide and stabilizers like 50% glycerol (pH 7.3-7.4) . Avoid repeated freeze-thaw cycles as this significantly reduces antibody performance .

How can researchers troubleshoot non-specific binding when using PMVK antibodies in Western blot applications?

Non-specific binding in Western blots can be addressed through several methodological approaches:

• Optimize blocking conditions: Test different blocking agents (5% non-fat milk, 5% BSA, commercial blockers) and blocking times (1-3 hours)

• Adjust antibody dilution: Start with the manufacturer's recommended range (e.g., 1:1000-1:4000) and titrate as needed

• Increase washing stringency: More frequent washes with higher concentrations of Tween-20 (0.1-0.3%)

• Pre-adsorb the antibody: Incubate with non-relevant tissue lysate before application to reduce cross-reactivity

• Use positive control lysates from cells known to express PMVK (HeLa, HepG2)

For PMVK-specific optimization, consider that it's a cytosolic protein, so nuclear-cytoplasmic fractionation might improve signal-to-noise ratio when investigating its expression in complex tissue samples .

What protocol modifications are recommended for optimal immunohistochemical detection of PMVK in different tissue types?

Immunohistochemical detection of PMVK requires careful optimization based on tissue type:

• Antigen retrieval: For most tissues, TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 is an alternative for some tissues

• Antibody dilution: Begin with 1:100-1:400 and adjust based on signal intensity and background

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

• Detection system: For tissues with lower PMVK expression, consider signal amplification methods such as polymer-based detection systems

PMVK shows differential expression across tissues, with highest levels in metabolically active organs like liver, heart, and kidney . When examining liver cancer tissues, be aware that PMVK expression may be altered compared to normal liver, potentially requiring adjusted protocols .

How can researchers quantitatively analyze PMVK expression levels using antibody-based methods?

Quantitative analysis of PMVK expression requires standardized approaches:

• Western blot densitometry:

  • Use loading controls appropriate for your experimental context (β-actin, GAPDH)

  • Establish a standard curve with recombinant PMVK protein

  • Ensure linear range detection by testing multiple exposure times or using digital imaging systems

• Quantitative immunohistochemistry:

  • Standardize staining conditions across all samples

  • Use digital image analysis software to quantify DAB intensity

  • Employ tissue microarrays when comparing multiple samples to minimize batch effects

• Flow cytometry quantification:

  • Use fluorescently-conjugated PMVK antibodies (e.g., Janelia Fluor® 635 conjugate)

  • Include calibration beads to standardize fluorescence intensity

  • Account for autofluorescence through proper controls

When comparing PMVK expression across different experimental conditions, always process and analyze samples in parallel to minimize technical variation .

How can PMVK antibodies be used to investigate the mevalonate pathway in metabolic disorders?

PMVK's role in the mevalonate pathway makes it valuable for metabolic disorder research:

• Use PMVK antibodies alongside other mevalonate pathway enzyme antibodies (HMG-CoA reductase, MVK) to assess pathway coordination

• Combine immunodetection of PMVK with metabolite analysis (e.g., mevalonate levels) to correlate enzyme expression with pathway activity

• Implement dual immunofluorescent staining to investigate co-localization of PMVK with sterol regulatory element-binding proteins (SREBPs)

Research has shown that PMVK is sterol-inducible, suggesting its expression may change in response to cellular cholesterol levels . This makes PMVK antibodies particularly useful for investigating disorders of cholesterol homeostasis and potential therapeutic interventions targeting the mevalonate pathway .

What emerging applications of PMVK antibodies are being developed for cancer research and diagnostics?

Emerging applications of PMVK antibodies in cancer research include:

• Prognostic biomarker development: Correlating PMVK expression with patient outcomes in various cancer types

• Therapeutic response prediction: Monitoring PMVK expression changes following treatment with statins or other mevalonate pathway inhibitors

• Companion diagnostics: Identifying patients likely to respond to emerging PMVK inhibitors like PMVKi5

• Combined analysis with microRNAs: Recent research has identified microRNA-874 as targeting PMVK and inhibiting cancer cell growth via the mevalonate pathway

The connection between PMVK and β-catenin signaling opens new avenues for investigating its role in cancers where Wnt/β-catenin signaling is dysregulated, such as colorectal cancer and hepatocellular carcinoma .

How should researchers design experiments to investigate the relationship between PMVK and CKI α in β-catenin regulation?

Based on recent discoveries linking PMVK to β-catenin regulation through CKI α, experiments should be designed to:

• Perform triple co-immunoprecipitation studies with antibodies against PMVK, β-catenin, and CKI α

• Use proximity ligation assays to visualize and quantify PMVK-CKI α interactions in situ

• Conduct domain mapping studies with truncated constructs to identify specific interaction regions

• Implement CRISPR-Cas9 gene editing to create PMVK mutants affecting key residues involved in protein-protein interactions

Research has shown that PMVK affects β-catenin signaling, potentially through interaction with CKI α . When designing these experiments, consider using both wild-type and mutant forms of these proteins to elucidate the structural basis of their interactions .

What experimental approaches are recommended for investigating the potential role of PMVK in embryonic development?

To investigate PMVK's role in embryonic development:

• Use PMVK antibodies for temporal expression profiling during different developmental stages

• Implement conditional knockout models with immunohistochemical validation of PMVK depletion

• Combine PMVK staining with markers of developmental pathways (Wnt/β-catenin, Hedgehog, Notch)

• Conduct rescue experiments with wild-type PMVK in knockout models to confirm specificity of developmental phenotypes

Research suggests PMVK is required for mouse embryonic development, with knockdown significantly reducing β-catenin signaling . The study of PMVK in development may provide insights into both normal developmental processes and developmental disorders associated with the mevalonate pathway or β-catenin signaling .

What best practices should researchers follow when reporting PMVK antibody-based experimental results in publications?

For reproducible research with PMVK antibodies, publications should include:

• Complete antibody information: manufacturer, catalog number, clone ID, lot number, RRID (Research Resource Identifier)

• Detailed methodological parameters: antibody dilution, incubation conditions, detection methods

• Validation evidence: western blot images showing single bands at expected molecular weight, positive and negative controls

• Quantification methods: software used, normalization procedures, statistical approaches

• Original unprocessed images as supplementary material

Including the citation format recommended by manufacturers (e.g., "Anti-Phosphomevalonate kinase PMVK Antibody Picoband® (Boster Biological Technology, Pleasanton CA, USA, Catalog # PA1067)") facilitates reagent tracking and experimental reproduction.