MVK Antibody

What is MVK and what is its biological function?

MVK (Mevalonate kinase) is a 42 kDa cytoplasmic protein that belongs to the GHMP kinase family. It catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, representing a critical early enzyme in isoprenoid and sterol synthesis pathways. The protein consists of 396 amino acids and plays a crucial role in cellular metabolism. Defects in MVK can lead to serious metabolic disorders including mevalonic aciduria (MEVA), which is characterized by an accumulation of mevalonic acid resulting in various symptoms such as psychomotor retardation, dysmorphic features, cataracts, hepatosplenomegaly, lymphadenopathy, anemia, hypotonia, myopathy, and ataxia .

What are the common types of MVK antibodies available for research?

MVK antibodies are available in several formats for research applications, including polyclonal and monoclonal variants. Polyclonal antibodies such as the 12228-1-AP are developed in rabbits using MVK fusion proteins as immunogens, while monoclonal antibodies like the 2C5 clone (H00004598-M02) are derived from mouse IgG2a Kappa. These antibodies come in various formats including unconjugated (most common), azide-free, and BSA-free preparations depending on the specific experimental requirements and research applications .

What are the main research applications for MVK antibodies?

MVK antibodies are primarily utilized in several key research applications:

These applications enable researchers to detect, localize, and quantify MVK protein in various biological samples, contributing to our understanding of its role in normal physiology and disease states .

How should I select the appropriate MVK antibody for my specific research application?

When selecting an MVK antibody, consider multiple factors that will impact experimental success: (1) First, evaluate the validated applications - different antibodies are optimized for specific techniques such as Western blot, IF/ICC, or ELISA. For example, the 12228-1-AP antibody has been validated for WB, IF/ICC, and ELISA applications, while H00004598-M02 is suitable for ELISA, IHC, and Western blot . (2) Species reactivity is essential - confirm that the antibody recognizes MVK in your experimental model (human, mouse, rat, etc.). (3) Consider the cellular localization requirements - cytoplasmic detection versus membrane-bound detection. (4) Review the literature for published applications of specific antibodies in similar experimental systems. (5) For quantitative studies, monoclonal antibodies may provide more consistent results due to their homogeneity, while polyclonal antibodies might offer higher sensitivity through recognition of multiple epitopes .

What controls should I include when using MVK antibodies in my experiments?

Implementing proper controls is essential for reliable MVK antibody experiments: (1) Positive controls should include samples known to express MVK, such as HepG2, A431, PC-3, or Jurkat cells as validated in the literature . (2) Negative controls should utilize samples where MVK expression is absent or has been knocked down via siRNA or CRISPR, such as the LV542-3 (MVK-1283) interference model described in published research . (3) For immunocytochemistry or immunohistochemistry, include a secondary antibody-only control to assess non-specific binding. (4) When performing knockdown/overexpression studies, include appropriate vector-only or scrambled controls as reference points. (5) For Western blots, blocking peptide controls can confirm antibody specificity by pre-incubating the antibody with its specific antigen before application to samples. These comprehensive controls will strengthen your data interpretation and address potential reviewer concerns regarding antibody specificity .

How do MVK antibody applications differ between human and animal model research?

MVK antibody applications show important species-specific considerations that researchers must address: (1) Cross-reactivity profiles vary significantly - while some antibodies like 12228-1-AP demonstrate reactivity with human, mouse, and rat MVK, others may have limited cross-reactivity . (2) Epitope conservation analysis is essential for cross-species applications - the amino acid sequence conservation between human MVK (UniProt ID: Q03426) and mouse MVK (UniProt ID: Q9R008) or rat MVK (UniProt ID: P17256) should be evaluated when transitioning between model systems . (3) Validation requirements differ - human-reactive antibodies require additional validation when applied to animal models, including Western blot confirmation at the expected molecular weight (42 kDa for MVK). (4) Application-specific optimizations may be necessary - dilution ranges for Western blot (1:1000-1:4000) or IF/ICC (1:10-1:100) often require species-specific adjustments. (5) For translational research spanning multiple species, selecting antibodies with demonstrated multi-species reactivity, such as those with published validation in both human and mouse models, will facilitate consistent experimental approaches .

How can MVK antibodies be used to study the relationship between MVK expression and cell differentiation?

MVK antibodies provide powerful tools for investigating the relationship between MVK expression and cell differentiation through multiple sophisticated approaches: (1) Immunoblotting combined with differentiation marker analysis - research has demonstrated that MVK interference decreases the expression of differentiation markers like keratin 1 and involucrin in keratinocytes, establishing connections between the mevalonate pathway and cellular differentiation programs . (2) Immunofluorescence co-localization studies with differentiation-stage specific markers can track MVK expression patterns during differentiation progression. (3) Chromatin immunoprecipitation (ChIP) assays using transcription factor antibodies combined with MVK expression analysis can elucidate regulatory mechanisms. (4) Time-course experiments tracking MVK expression during differentiation using quantitative Western blot and IF can establish temporal relationships. (5) Rescue experiments using MVK overexpression in differentiation-defective cells, followed by immunodetection of both MVK and differentiation markers, can establish causality. Published research demonstrates that decreased keratin 1 and involucrin expression following MVK interference was significantly attenuated by farnesyl pyrophosphate (FPP) supplementation, highlighting the metabolic connections between MVK activity and differentiation processes .

What methodological approaches can be used to study the role of MVK in protein prenylation using MVK antibodies?

Investigating MVK's role in protein prenylation requires sophisticated methodological approaches: (1) Immunoprecipitation combined with prenylation-specific detection methods - MVK can be immunoprecipitated from cell lysates using specific antibodies (4 μg of anti-MVK antibody is typically sufficient for 400 μg of cell lysate) . (2) Dual immunofluorescence labeling with MVK antibodies and prenylated protein markers can visualize spatial relationships. (3) Western blot analysis comparing prenylated protein levels in MVK-deficient versus normal cells reveals functional impacts - research has demonstrated that MVK interference decreases protein prenylation levels, which can be rescued by geranylgeranyl pyrophosphate (GGPP) supplementation . (4) Pulse-chase experiments with radioactive prenylation precursors, followed by MVK immunoprecipitation, can track the kinetics of the process. (5) Advanced proteomics approaches combining MVK antibody-based enrichment with mass spectrometry can identify novel prenylated protein targets. This methodological framework allows researchers to comprehensively characterize how MVK regulates protein prenylation and subsequent cellular functions, with evidence showing that defective protein prenylation serves as a diagnostic biomarker of MVK deficiency .

How can MVK antibodies be applied in studying the relationship between MVK deficiency and apoptosis?

MVK antibodies enable multifaceted investigation of the MVK deficiency-apoptosis relationship: (1) Immunoblotting combined with apoptotic marker detection - research has demonstrated that MVK interference significantly increases apoptotic rates in cell culture models, which can be attenuated by GGPP supplementation but not by FPP, suggesting pathway-specific regulatory mechanisms . (2) Immunofluorescence co-staining with MVK antibodies and apoptotic markers (cleaved caspases, TUNEL) in MVK-deficient tissues provides spatial context. (3) Flow cytometry coupling MVK staining with annexin V/propidium iodide allows quantitative single-cell analysis of the relationship. (4) Proximity ligation assays between MVK and apoptotic regulatory proteins can reveal direct molecular interactions. (5) In vivo models of MVK deficiency analyzed using tissue immunohistochemistry with MVK antibodies and apoptotic markers can translate findings to physiological contexts. Research findings indicate that MVK overexpression significantly decreases apoptotic rates in human keratinocytes, while MVK interference increases apoptosis through mechanisms potentially involving decreased protein prenylation levels, as thienopyrimidine-based bisphosphonate inhibitors of GGPP synthesis block protein prenylation and subsequently trigger cellular apoptosis in multiple myeloma cells .

What are the optimal protocols for using MVK antibodies in Western blot applications?

For optimal Western blot results with MVK antibodies, follow this comprehensive protocol: (1) Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors; HepG2, A431, PC-3, and Jurkat cells are validated positive controls expressing detectable MVK levels . (2) Protein separation: Load 20-40 μg protein per lane on 10-12% SDS-PAGE gels for optimal separation near the 42 kDa marker (MVK's observed molecular weight). (3) Transfer: Use PVDF membrane for optimal protein binding and subsequently block with 5% non-fat milk in TBST for 1 hour at room temperature. (4) Primary antibody incubation: Dilute anti-MVK antibody at 1:1000-1:4000 in blocking buffer and incubate overnight at 4°C; for example, 0.1 μg/mL concentration has been validated for HepG2, A431, and HEK001 cell lysates . (5) Secondary antibody: Incubate with HRP-conjugated secondary antibody (anti-rabbit for polyclonal antibodies like 12228-1-AP, anti-mouse for monoclonal antibodies) at 1:2000-1:5000 dilution for 1 hour at room temperature. (6) Detection: Use enhanced chemiluminescence reagents and expose to film or digital imager; expect a specific band at approximately 42 kDa. (7) Validation: Consider using reducing conditions with Immunoblot Buffer Group 1 for optimal results, as specified in published protocols .

What is the recommended protocol for immunofluorescence staining using MVK antibodies?

For successful immunofluorescence detection of MVK protein, implement this validated protocol: (1) Cell preparation: Culture cells (PC-3 or A431 cells are recommended based on validation data) on coverslips or chamber slides to 70-80% confluence . (2) Fixation: Immersion-fix cells using 4% paraformaldehyde for 10-15 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes. (3) Blocking: Incubate with 5-10% normal serum (matching the species of the secondary antibody) in PBS for 30-60 minutes. (4) Primary antibody: Dilute MVK antibody at 1:10-1:100 in blocking solution and incubate for 3 hours at room temperature or overnight at 4°C; successful staining has been achieved with 2 μg/mL concentration in A431 cells . (5) Secondary antibody: After washing, apply fluorophore-conjugated secondary antibody such as NorthernLights 557-conjugated Anti-Rabbit IgG (for polyclonal antibodies) at manufacturer's recommended dilution for 1 hour at room temperature. (6) Counterstaining: Use DAPI (blue) nuclear counterstain for proper cellular localization context. (7) Mounting and imaging: Mount with anti-fade medium and observe under fluorescence microscope; expect cytoplasmic localization of MVK based on published results. This protocol has successfully demonstrated specific cytoplasmic staining of MVK in multiple cell types .

What are the considerations for using MVK antibodies in immunoprecipitation experiments?

For successful immunoprecipitation of MVK protein, implement these critical protocol elements: (1) Sample preparation: Prepare cell lysates in a non-denaturing lysis buffer (such as NP-40 or CHAPS-based buffers) containing protease inhibitors; HepG2 and A431 cells are recommended based on validated results . (2) Antibody amount optimization: Use 4 μg of purified anti-MVK antibody per 400 μg of cell lysate for effective immunoprecipitation, as validated in published protocols . (3) Binding substrate selection: Couple the antibody to Protein A/G-coated plates (for polyclonal rabbit antibodies) or appropriate beads; plate-based immunoprecipitation has been successfully performed using 4 wells of a 96-well Corning Costar EIA/RIA plate . (4) Incubation conditions: Allow antibody-antigen binding to occur for 2 hours at room temperature with gentle agitation to maintain protein conformation. (5) Washing stringency: Perform 3-5 washes with lysis buffer to remove non-specific interactions while preserving specific MVK binding. (6) Elution method: Use either acidic elution (pH 2.5-3.0) followed by immediate neutralization, or direct elution in SDS-PAGE sample buffer for subsequent Western blot analysis. (7) Detection: Perform Western blot under reducing conditions using 0.1 μg/mL anti-MVK antibody to confirm successful immunoprecipitation of the 42 kDa MVK protein. This approach has been validated for detecting both endogenous MVK from cell lysates and recombinant human MVK .

What are common issues when using MVK antibodies and how can they be resolved?

When working with MVK antibodies, researchers often encounter these challenges with corresponding solutions: (1) Weak or no signal in Western blots can be addressed by increasing protein loading (40-60 μg/lane), optimizing antibody concentration (try a range between 1:500-1:4000), extending primary antibody incubation to overnight at 4°C, and using enhanced chemiluminescence detection systems . (2) High background in immunostaining requires more stringent blocking (5-10% serum with 0.1-0.3% Triton X-100), increased wash duration (5-6 washes of 5-10 minutes each), and optimized antibody dilution (test range from 1:10-1:100) . (3) Multiple bands in Western blot can be resolved by using freshly prepared samples with complete protease inhibitors, adjusting reducing agent concentration, and confirming positive control samples show the expected 42 kDa band . (4) Poor immunoprecipitation efficiency can be improved by increasing antibody amount (up to 6 μg per 400 μg lysate), extending incubation time (up to 4 hours), and using optimized lysis buffers that preserve native protein conformation . (5) Cross-reactivity issues can be addressed by pre-absorbing the antibody with non-target species proteins or selecting antibodies with validated species specificity for your experimental system .

How can I validate that my MVK antibody is specifically detecting MVK protein in my experimental system?

Comprehensive MVK antibody validation requires multiple complementary approaches: (1) Positive and negative control samples - use cell lines with confirmed MVK expression (HepG2, A431, PC-3, Jurkat cells) as positive controls, and implement MVK knockdown models (such as LV542-3/MVK-1283 interference) as negative controls . (2) Molecular weight verification - confirm detection at the expected 42 kDa size in Western blots using protein ladders . (3) Peptide competition assays - pre-incubate the antibody with excess immunizing peptide to confirm signal reduction in true positive samples. (4) Orthogonal method comparison - correlate protein detection with MVK mRNA levels using RT-qPCR in the same samples. (5) Multiple antibody validation - compare staining patterns between two antibodies targeting different MVK epitopes. (6) Functional validation - demonstrate that phenotypic effects of MVK knockdown (such as decreased differentiation marker expression or increased apoptosis) correlate with reduced MVK protein detection . (7) Overexpression systems - transfect cells with MVK expression vectors and confirm increased signal intensity at the correct molecular weight, as demonstrated in published MVK overexpression studies .

How should I interpret conflicting results when studying MVK expression in different cell types or experimental conditions?

When encountering conflicting MVK expression data, apply this systematic interpretive framework: (1) Technical vs. biological variation analysis - determine whether discrepancies arise from technical limitations (antibody sensitivity, protocol differences) or represent true biological differences by standardizing detection methods across experiments . (2) Cell type-specific expression patterns - recognize that MVK expression varies considerably across cell types; HepG2, A431, PC-3, and Jurkat cells show consistently detectable expression, while other cell types may exhibit contextual expression . (3) Post-translational modification effects - consider that MVK may undergo modifications affecting epitope accessibility or apparent molecular weight in different cellular contexts. (4) Sample preparation impact - different lysis methods may preferentially extract MVK from distinct cellular compartments, influencing detection. (5) Threshold interpretation - establish quantitative thresholds for "positive" MVK expression based on validated controls. (6) Literature contextualization - interpret your findings within the existing knowledge framework; for example, research has demonstrated that MVK interference decreases differentiation marker expression (keratin 1 and involucrin), which can be rescued by FPP supplementation, providing mechanistic context for seemingly contradictory results . (7) Experimental condition relevance - factors like cell confluence, growth phase, and media components can significantly impact MVK expression and should be standardized when comparing across experimental conditions.

How can MVK antibodies be utilized to study MVK deficiency disorders?

MVK antibodies provide critical tools for investigating MVK deficiency disorders through multiple research approaches: (1) Diagnostic biomarker development - MVK protein levels detected by immunoblotting can be correlated with clinical presentations of mevalonic aciduria (MEVA) and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS) . (2) Genotype-phenotype correlation studies - using MVK antibodies to quantify protein expression in patient-derived samples with different MVK mutations can reveal how specific genetic variants impact protein levels and stability. (3) Functional characterization - comparing prenylation levels between normal and MVK-deficient samples using immunodetection methods can elucidate pathophysiological mechanisms, as defective protein prenylation has been established as a diagnostic biomarker of MVK deficiency . (4) Therapeutic screening platforms - MVK antibodies enable high-throughput screening of compounds that might stabilize mutant MVK protein or restore its function by monitoring protein levels in treated vs. untreated patient cells. (5) Animal model validation - immunohistochemical analysis of MVK expression in transgenic disease models allows translation between animal and human pathophysiology. This comprehensive approach facilitates both mechanistic understanding and potential therapeutic advancement for these rare but serious metabolic disorders .

What is the potential for using MVK as a therapeutic target, and how can antibodies facilitate this research?

MVK represents a promising therapeutic target with antibody-based research applications: (1) Target validation studies using MVK antibodies have established connections between MVK activity and critical cellular processes including differentiation and apoptosis regulation . (2) Rescue experiment design - MVK antibodies enable monitoring of protein restoration following interventions; research demonstrates that FPP can rescue differentiation defects while GGPP can attenuate apoptosis in MVK-deficient models, providing pathway-specific therapeutic insights . (3) Drug discovery screening - high-content screening platforms using MVK antibody-based detection can identify compounds that modulate MVK expression or stability. (4) Therapeutic protein delivery monitoring - tracking exogenously delivered MVK protein using antibody detection in enzyme replacement therapy approaches. (5) Immunotherapy potential - while direct antibody therapeutics may not apply to cytoplasmic targets like MVK, antibody-drug conjugates could potentially deliver modulators to cells with abnormal MVK expression. (6) Biomarker development - MVK antibodies facilitate the development of companion diagnostics to identify patients likely to respond to pathway-targeted therapies. These applications highlight MVK's therapeutic relevance across multiple conditions including metabolic disorders, inflammatory diseases, and potentially cancer contexts where isoprenoid pathway dysregulation occurs .

What emerging techniques might enhance MVK antibody applications in research?

Innovative methodological approaches are expanding MVK antibody applications: (1) Super-resolution microscopy combined with MVK antibody detection can reveal previously unobservable subcellular localization patterns and protein interactions at nanometer resolution. (2) Single-cell proteomics incorporating MVK antibodies enables heterogeneity analysis across cell populations, potentially revealing functional subpopulations with distinct MVK expression patterns. (3) Spatial transcriptomics combined with MVK immunodetection can correlate protein levels with transcriptional programs in specific tissue microenvironments. (4) Antibody engineering approaches, including the development of recombinant antibody fragments with enhanced tissue penetration or intracellular delivery capabilities, may overcome current limitations in accessing MVK in complex samples. (5) Mass cytometry (CyTOF) incorporating metal-conjugated MVK antibodies allows simultaneous detection of MVK alongside dozens of other markers in single cells. (6) CRISPR-based genetic screens coupled with MVK antibody detection can systematically identify genes that regulate MVK expression, stability, or activity. (7) Artificial intelligence-assisted image analysis of MVK immunostaining patterns may reveal subtle phenotypes and correlations not detectable by conventional analysis methods. These emerging technologies will significantly expand our understanding of MVK biology in both normal physiology and disease states .

How might MVK research contribute to our understanding of broader metabolic pathways and disease mechanisms?

MVK research offers significant insights into fundamental biological processes and disease mechanisms: (1) Isoprenoid pathway regulation - MVK antibody-based research has demonstrated connections between the mevalonate pathway and cell differentiation, with MVK interference decreasing expression of keratin 1 and involucrin, highlighting the pathway's significance beyond cholesterol synthesis . (2) Protein prenylation mechanisms - studies show that MVK interference decreases prenylation levels, which can be rescued by GGPP supplementation, establishing MVK as a key regulator of this critical post-translational modification system . (3) Cellular stress response integration - MVK's involvement in apoptosis regulation suggests it functions as a metabolic checkpoint linking isoprenoid availability to cell survival decisions . (4) Inflammatory disorder mechanisms - MVK's role in periodic fever syndromes bridges metabolism and immunity, potentially informing therapeutic approaches for other inflammatory conditions. (5) Cancer metabolism connections - the mevalonate pathway's importance in rapidly dividing cells suggests MVK may represent an unexplored metabolic vulnerability in certain cancers. (6) Developmental biology implications - the differentiation defects observed with MVK interference suggest developmental roles that may explain some symptoms of MVK deficiency disorders . (7) Therapeutic target identification - understanding MVK regulatory networks may reveal additional drug targets within connected metabolic pathways. These multifaceted insights demonstrate how focused MVK research contributes to our understanding of integrative cellular metabolism and disease mechanisms .