MLX Antibody

What is MLX protein and why is it important in cellular research?

MLX (Max-like protein X) functions as a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that plays a crucial role in regulating gene expression related to cell growth, proliferation, and apoptosis . It primarily localizes in the nucleus where it forms either homodimers or heterodimers with Myc family proteins (including Myc, Mad1, Mad3, Mad4, Mxi1, and Mnt) to bind to the E-box sequence CACGTG in DNA . This interaction is fundamental for the regulation of target genes that influence cellular processes and maintain homeostasis . MLX also forms important heterodimers with Mondo family proteins, especially MondoA, which creates a functional transcriptional complex involved in nutrient sensing and metabolic regulation . The significance of MLX in research stems from its central position in transcriptional networks and its implications in various cellular processes and disease states, including its association with Williams-Beuren syndrome through its interaction with WBSCR14 .

What applications are MLX antibodies suitable for in research settings?

MLX antibodies are versatile tools suitable for multiple research applications. Based on validated antibody products, MLX antibodies can be effectively used in western blotting (WB) at dilutions ranging from 1:500-1:2000, immunohistochemistry (IHC) at 1:100-1:300, immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) at approximately 1:10000 dilution . These applications enable researchers to detect and quantify MLX protein expression, examine its subcellular localization, and study its interactions with other proteins. The availability of MLX antibodies in various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, further expands their research utility across different experimental platforms and detection systems .

How should MLX antibodies be stored and handled to maintain optimal activity?

For optimal preservation of MLX antibody activity, proper storage and handling are essential. Long-term storage should be at -20°C, which maintains antibody stability for up to one year . For frequent use and short-term storage (up to one month), refrigeration at 4°C is recommended to reduce the stress of freeze-thaw cycles . It's critical to avoid repeated freeze-thaw cycles as these can significantly compromise antibody functionality . Most commercial MLX antibodies are supplied in stabilizing buffers that typically contain PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide to enhance antibody stability . When working with MLX antibodies, follow standard antibody handling practices: briefly centrifuge vials before opening, use sterile technique when aliquoting, and handle at appropriate temperatures as specified by the manufacturer to maintain binding affinity and specificity across experimental applications.

What are the key characteristics of commonly used MLX antibodies?

Commercially available MLX antibodies present diverse characteristics that researchers should consider when selecting the appropriate reagent for their experiments. The table below summarizes key features of typical MLX antibodies:

These antibodies recognize MLX protein across multiple species, with the mouse monoclonal F-12 antibody being particularly well-characterized for detecting MLX in human, mouse, and rat samples across various applications . The rabbit polyclonal antibody provides flexibility for different applications, especially for immunohistochemistry where it has been validated on human brain tissue samples .

How can researchers investigate MLX-MondoA complex formation using appropriate antibody techniques?

To investigate MLX-MondoA complex formation, researchers should implement a multi-technique approach centered on co-immunoprecipitation (co-IP) followed by complementary validation methods. Begin with co-IP using an MLX antibody (such as mouse monoclonal F-12) conjugated to agarose beads to pull down MLX and its interacting partners from cell lysates . Follow with western blotting to detect MondoA in the immunoprecipitated complex, using appropriate MondoA-specific antibodies . This approach can effectively capture the enhanced binding observed between MondoA and mutant variants like MLX-Q139R compared to wild-type MLX .

To further characterize the functional consequences of MLX-MondoA interaction, implement chromatin immunoprecipitation (ChIP) assays using MLX antibodies to assess binding to target promoters, such as TXNIP . This can reveal how mutations affect DNA binding efficiency of the complex. Complement these biochemical approaches with proximity ligation assays (PLA) or Förster resonance energy transfer (FRET) using fluorescently labeled antibodies to visualize and quantify MLX-MondoA interactions in intact cells. Additionally, reporter assays with MLX-regulated promoters (like TXNIP) can assess the transcriptional impact of this interaction . Together, these methods provide a comprehensive assessment of both physical association and functional consequences of MLX-MondoA complex formation.

What strategies should be employed to study the subcellular localization of MLX and its translocation between cytoplasm and nucleus?

Studying MLX subcellular localization requires a systematic approach combining immunofluorescence microscopy with biochemical fractionation. Begin with immunofluorescence using validated MLX antibodies (working dilution 1:100-1:300) and high-resolution confocal microscopy to visualize the baseline distribution of MLX in fixed cells . Co-stain with nuclear markers (DAPI/Hoechst) and cytoplasmic markers to establish reference points. For dynamic studies of MLX translocation in response to stimuli (such as leptomycin B, which can trigger nuclear translocation of the MondoA/MLX complex), implement live-cell imaging using cells expressing fluorescently tagged MLX constructs .

Complement imaging with subcellular fractionation followed by western blotting to quantitatively assess MLX distribution between nuclear and cytoplasmic compartments under various conditions. For more precise analysis, perform chromatin immunoprecipitation (ChIP) to measure MLX binding to target DNA sequences under different cellular conditions . When investigating MLX localization to specific subcellular structures such as lipid droplets, employ co-localization studies with lipid droplet-specific dyes or markers, as MLX has been shown to bind to lipid droplets through its C-terminal amphipathic helix . For comprehensive assessment, implement proximity-based proteomic approaches such as BioID or APEX to identify proteins in close proximity to MLX in different cellular compartments, providing insights into compartment-specific interaction networks.

How does the MLX-Q139R mutation affect experimental outcomes in inflammation and autophagy studies?

The MLX-Q139R mutation (rs665268) significantly impacts experimental outcomes in inflammation and autophagy studies through multiple mechanisms that researchers should carefully consider. This mutation enhances MLX-MondoA heterodimer formation and increases binding to target DNA sequences such as the TXNIP promoter, resulting in upregulated TXNIP expression . When designing experiments, researchers should anticipate that cells expressing MLX-Q139R will show enhanced inflammasome formation and attenuated autophagy compared to those expressing wild-type MLX .

In experimental systems, the Q139R mutation produces several measurable effects: (1) increased TXNIP protein levels detectable by western blotting, (2) enhanced NLRP3 inflammasome formation, (3) decreased numbers of autophagosomes observable by electron microscopy, (4) reduced LC3II expression with concurrent upregulation of p62, and (5) increased expression of LARS1, mTOR, and phospho-mTOR (Ser2448) . When conducting lipopolysaccharide-induced inflammation studies, researchers should adjust experimental parameters to account for the differential response between wild-type and Q139R-expressing cells. For comprehensive assessment, implement assays measuring both inflammasome activity (IL-1β secretion, caspase-1 activation) and autophagy markers (LC3II/I ratio, p62 levels, autophagic flux) to capture the dual impact of this mutation on inflammatory and autophagic processes.

What are the challenges in detecting MLX binding to lipid droplets and how can they be overcome?

Detecting MLX binding to lipid droplets presents several technical challenges that require specialized approaches. The C-terminal amphipathic helix of MLX mediates its binding to lipid droplets , but this interaction may be dynamic or context-dependent, complicating consistent detection. To overcome these challenges, researchers should implement a multi-modal strategy combining optimized immunofluorescence with biochemical validation.

For immunofluorescence, use permeabilization conditions that preserve lipid droplet integrity (avoiding strong detergents) while allowing antibody access. Co-stain with lipid droplet-specific dyes (BODIPY 493/503 or LipidTOX) and perform high-resolution confocal or super-resolution microscopy to precisely visualize co-localization. For biochemical validation, isolate lipid droplet fractions using sucrose gradient ultracentrifugation and confirm MLX presence using western blotting with sensitive detection methods.

To address potential challenges with antibody accessibility to MLX epitopes when bound to lipid droplets, consider using epitope tags (His, FLAG, HA) on recombinant MLX constructs expressed in cellular systems, which can provide consistent detection regardless of protein conformation. For dynamic studies, implement fluorescence recovery after photobleaching (FRAP) or photoactivatable-GFP tagged MLX to measure binding kinetics to lipid droplets under various metabolic conditions. Additionally, construct MLX mutants with alterations in the C-terminal amphipathic helix to serve as negative controls for lipid droplet binding specificity. Finally, use proximity labeling techniques such as APEX2 fused to MLX to identify neighboring proteins at the lipid droplet surface, providing a comprehensive map of the MLX interactome in this specific subcellular location.

What optimization steps are necessary for using MLX antibodies in western blotting?

Successful western blotting with MLX antibodies requires systematic optimization across multiple parameters. Begin with sample preparation: use RIPA or NP-40 based lysis buffers containing protease inhibitors to effectively extract MLX protein while preserving its integrity. Given MLX's calculated molecular weight of approximately 33.3 kDa , use 10-12% polyacrylamide gels for optimal resolution around this size range. For primary antibody incubation, test a range of dilutions (1:500-1:2000 as recommended) to determine optimal signal-to-noise ratio for your specific sample type and detection system.

Blocking conditions significantly impact results—try both 5% BSA and 5% non-fat dry milk in TBST to determine which provides minimal background with your antibody. For membrane transfer, use PVDF membranes which generally provide better protein retention and signal strength for transcription factors like MLX. When validating specificity, include positive controls (cell lines with known MLX expression) and negative controls (MLX knockdown samples or competing peptide blocks). If detecting specific MLX variants or mutations (such as Q139R), validate antibody recognition using overexpression systems with wild-type and mutant constructs. For challenging samples with low MLX expression, consider signal amplification methods such as HRP-conjugated polymer detection systems or implement a biotin-streptavidin detection strategy using biotinylated secondary antibodies for enhanced sensitivity.

How should researchers design immunoprecipitation experiments to study MLX protein interactions?

Designing effective immunoprecipitation (IP) experiments for MLX protein interactions requires careful consideration of multiple factors. Begin by selecting an appropriate MLX antibody with validated IP performance, such as the mouse monoclonal F-12 antibody, which is available in agarose-conjugated form specifically optimized for IP applications . For lysis conditions, use gentle non-ionic detergent buffers (0.5-1% NP-40 or Triton X-100) with physiological salt concentrations (150mM NaCl) to preserve native protein interactions, particularly important for studying MLX-MondoA complexes .

Pre-clear lysates with appropriate control beads to reduce non-specific binding, and include negative controls (isotype-matched control antibodies or IgG) in parallel experiments. When investigating specific interactions like MLX-MondoA, include additional controls such as cells expressing MLX mutants (e.g., Q139R) to assess how mutations affect interaction strength . For co-IP experiments, use reciprocal approaches (IP with MLX antibody and probe for MondoA, then IP with MondoA antibody and probe for MLX) to robustly validate interactions.

To preserve transient or weak interactions, consider using chemical crosslinking (1-2% formaldehyde for 10 minutes) before cell lysis or include stabilizing agents in lysis buffers. For enhanced sensitivity when detecting interaction partners, consider tandem approaches like IP followed by mass spectrometry rather than just western blotting. This approach can reveal novel interaction partners beyond those targeted by specific antibodies. Finally, validate key interactions identified through IP using orthogonal methods such as proximity ligation assays or FRET to confirm their biological relevance in intact cellular contexts.

What controls are essential when performing immunohistochemistry with MLX antibodies?

When performing immunohistochemistry (IHC) with MLX antibodies, implementing robust controls is crucial for generating reliable and interpretable results. Include positive control tissues with known MLX expression patterns—human brain tissue has been validated for MLX antibody staining at 1:100 dilution . Include negative controls by omitting primary antibody while maintaining all other staining steps to assess potential background from secondary antibodies or detection systems. For additional specificity validation, use antigen blocking peptides (where available) to confirm signal specificity, or tissues from MLX knockout models if accessible.

Technical controls should include standardization of fixation protocols, as overfixation can mask epitopes while underfixation may compromise tissue morphology. Implement antigen retrieval optimization—test both heat-induced epitope retrieval methods (citrate buffer, pH 6.0 and EDTA buffer, pH 9.0) to determine which best exposes MLX epitopes in your tissue type. For antibody validation, perform dilution series (beginning with the recommended 1:100-1:300 range) to identify the optimal concentration that maximizes specific signal while minimizing background.

When examining altered MLX expression in disease contexts (such as Takayasu arteritis associated with the Q139R mutation) , include tissues from both normal and disease states for comparative analysis. If multiple antibodies against different MLX epitopes are available, use them in parallel on serial sections to cross-validate staining patterns. For chromogenic IHC, include isotype-matched control antibodies on adjacent sections. For fluorescent multiplex IHC, include single-color controls to assess spectral overlap and implement appropriate compensation. Finally, validate key IHC findings with orthogonal techniques such as in situ hybridization for MLX mRNA or western blotting of tissue lysates to corroborate protein expression levels.

How can researchers quantitatively assess MLX-regulated gene expression changes?

Quantitative assessment of MLX-regulated gene expression requires a multi-tiered approach combining promoter analysis, expression profiling, and functional validation. Begin with reporter assays using luciferase constructs containing MLX-responsive promoters such as TXNIP . This approach enables direct measurement of transcriptional activity and can reveal how mutations (like Q139R) affect MLX function. In these assays, co-transfect cells (such as human aortic smooth muscle cells) with MondoA and either wild-type or mutant MLX to assess differential effects on promoter activation .

For genome-wide analysis of MLX-regulated genes, implement RNA-sequencing of cells with MLX knockdown, overexpression, or mutation (Q139R), compared to appropriate controls. Follow with pathway enrichment analysis to identify biological processes affected by MLX activity. Validate key expression changes using quantitative RT-PCR, focusing on established MLX target genes like TXNIP and evaluating how perturbations affect their expression levels.

To directly assess MLX binding to target promoters, perform chromatin immunoprecipitation (ChIP) with MLX antibodies followed by qPCR of specific promoter regions (ChIP-qPCR) or sequencing (ChIP-seq) for genome-wide binding profiles . This approach can reveal how mutations like Q139R enhance DNA binding capacity . For functional validation, implement CRISPR-Cas9 gene editing to introduce specific MLX mutations in cellular models, then assess resulting phenotypes in relevant processes like autophagy (measuring LC3II/I ratios, p62 levels) or inflammasome activation (IL-1β secretion, caspase-1 activation) . Finally, for translational relevance, analyze tissue samples from patients with MLX mutations (such as rs665268/Q139R associated with Takayasu arteritis) to correlate genotype with disease-specific gene expression patterns .

What are common issues when working with MLX antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with MLX antibodies that can impact experimental outcomes. One common issue is weak or absent signal in western blotting despite confirmed MLX expression. This may result from inefficient protein extraction, as MLX can shuttle between the nucleus and cytoplasm depending on cellular conditions . To resolve this, use whole cell lysates prepared with buffer containing both non-ionic detergents (for membrane proteins) and high salt (>300mM NaCl) for nuclear extraction. Additionally, optimize transfer conditions for proteins around MLX's molecular weight (~33.3 kDa) by using semi-dry transfer with methanol-containing buffer.

Another frequent problem is non-specific bands in western blots. Address this by implementing more stringent blocking (5% BSA instead of milk, which contains bioactive proteins) and washing conditions (0.1% Tween-20 in TBS with 500mM NaCl). If high background persists in immunofluorescence or IHC, pre-absorb antibodies with cell/tissue lysates from species matching your experimental system but lacking the target protein.

For inconsistent immunoprecipitation results, particularly when studying MLX-MondoA interactions, optimize lysis conditions to preserve protein complexes. Use gentler detergents (0.5% NP-40) and include protease inhibitors along with phosphatase inhibitors, as phosphorylation status may affect complex formation. If epitope masking occurs due to protein-protein interactions (particularly in the MondoA-MLX complex), try alternative antibodies targeting different MLX epitopes or use epitope-tagged recombinant versions of MLX. For all applications, validate antibody specificity using MLX knockout or knockdown controls, especially when investigating mutant variants like Q139R .

How should researchers interpret conflicting results between different antibody-based assays for MLX?

When faced with conflicting results across different antibody-based assays for MLX, researchers should implement a systematic analytical approach. First, consider epitope accessibility differences between applications: in western blotting, denatured proteins expose all linear epitopes, while in applications with native proteins (IP, IF, IHC), tertiary structure may mask certain epitopes . This is particularly relevant for MLX, which forms complexes with partners like MondoA that could obscure antibody binding sites .

Evaluate assay-specific technical factors that might contribute to discrepancies. For instance, fixation conditions in IHC or IF may alter epitope recognition compared to non-fixed samples in western blotting. Cross-reactive proteins with similar molecular weights might be detected in western blots but distinguished by localization in microscopy-based methods. To resolve these conflicts, implement orthogonal validation approaches: use multiple antibodies targeting different epitopes of MLX, combine antibody-based methods with non-antibody techniques (such as mass spectrometry for protein identification), or utilize genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity.

For functional studies, particularly when investigating the effects of mutations like Q139R on MLX function, discrepancies might reflect genuine biological complexity rather than technical artifacts . In these cases, cellular context matters significantly—MLX functions differently depending on cell type, metabolic state, and expression levels of binding partners like MondoA. Document experimental conditions thoroughly, including cell density, serum conditions, and time points, as these factors may influence MLX localization and function. Finally, when publishing conflicting results, transparently report all findings along with detailed methodological information to allow the scientific community to interpret and build upon the complexity of MLX biology.

What statistical approaches are appropriate for analyzing MLX expression data across different experimental conditions?

The appropriate statistical analysis of MLX expression data requires tailored approaches based on experimental design and data characteristics. For comparing MLX expression levels across multiple experimental conditions (such as wild-type vs. Q139R mutation in different cell types) , begin with normality testing (Shapiro-Wilk test) to determine if parametric or non-parametric tests are appropriate. For normally distributed data comparing two conditions, use Student's t-test (paired or unpaired depending on experimental design); for multiple conditions, implement one-way ANOVA followed by appropriate post-hoc tests (Tukey's or Dunnett's depending on comparison structure).

When analyzing western blot densitometry data for MLX expression, normalize to appropriate loading controls (β-actin, GAPDH) and present data as fold change relative to control conditions with clear indication of sample size and variance measures. For time-course experiments examining MLX translocation or expression changes, repeated measures ANOVA or mixed-effects models may be more appropriate than multiple individual t-tests to control for family-wise error rate.

For complex experimental designs investigating how MLX mutations affect multiple downstream targets simultaneously (such as TXNIP, LARS1, mTOR, and autophagy markers) , consider multivariate approaches such as MANOVA or principal component analysis to account for correlations between related outcome measures. When examining the association between MLX genotypes (like rs665268) and clinical parameters in patient cohorts , implement appropriate statistics for genetic association studies, including odds ratios with confidence intervals, chi-square tests for categorical outcomes, and regression models for continuous variables with adjustment for relevant covariates. For all analyses, report effect sizes alongside p-values to convey biological significance beyond statistical significance, and consider implementing false discovery rate correction when performing multiple comparisons to control for type I errors.

How can MLX antibodies be utilized in studying metabolic regulation and nutrient sensing?

MLX antibodies offer powerful tools for investigating metabolic regulation and nutrient sensing through several advanced applications. The MondoA-MLX complex functions as a nutrient-responsive transcription factor system, particularly sensitive to glucose levels through regulation of TXNIP expression . Researchers can implement chromatin immunoprecipitation (ChIP) using MLX antibodies to map genome-wide binding patterns under various metabolic conditions (glucose starvation, excess, or alternative carbon sources), revealing condition-specific regulatory mechanisms. Combined ChIP-seq and RNA-seq approaches can correlate MLX binding events with transcriptional outcomes across the metabolome.

For dynamic studies of nutrient sensing, researchers should use immunofluorescence with MLX antibodies to track nuclear-cytoplasmic shuttling in response to metabolic stimuli, as the MondoA/MLX complex translocates to the nucleus under specific conditions . Time-course experiments combining subcellular fractionation with western blotting can quantify this translocation kinetics. The discovery that MLX binds to lipid droplets through its C-terminal amphipathic helix opens avenues for investigating how this localization affects metabolic sensing—researchers can use co-immunofluorescence with lipid droplet markers to assess how nutrient status affects this distribution.

For functional metabolic studies, combine MLX antibodies with metabolic flux analysis (Seahorse XF analyzer) to correlate MLX activity with changes in glycolysis, oxidative phosphorylation, and substrate utilization patterns. Additionally, proximity-based proteomic approaches (BioID, APEX) using MLX as bait can identify condition-specific interaction partners under different metabolic states, revealing how the MLX interactome adapts to nutrient availability. These approaches collectively provide a comprehensive understanding of MLX's role in integrating metabolic signals with transcriptional responses.

What role might MLX antibodies play in investigating autoimmune conditions like Takayasu arteritis?

MLX antibodies represent critical tools for investigating the pathogenesis of Takayasu arteritis (TAK) and potentially other autoimmune conditions, particularly given the significant association between the MLX rs665268 polymorphism (Q139R) and TAK development . Researchers can implement immunohistochemistry using MLX antibodies on vascular tissue specimens from TAK patients to assess MLX expression patterns and localization, particularly in arterial walls and inflammatory infiltrates. This approach can help determine whether MLX expression correlates with disease severity or specific clinical manifestations such as aortic regurgitation, which has been associated with the MLX-GG risk genotype .

To investigate the molecular mechanisms underlying this association, implement co-immunoprecipitation with MLX antibodies followed by mass spectrometry to identify differential protein interactions between wild-type MLX and the Q139R variant in relevant cell types like vascular smooth muscle cells or immune cells from patients. Complement this with ChIP-seq using MLX antibodies to map genome-wide binding differences between MLX variants, potentially revealing dysregulated target genes contributing to autoimmunity.

Since the Q139R mutation enhances MLX-MondoA complex formation and increases TXNIP expression while attenuating autophagy and enhancing inflammasome formation , researchers should use MLX antibodies in multiplex immunofluorescence to simultaneously visualize MLX, inflammasome components (NLRP3, ASC, caspase-1), and autophagy markers (LC3, p62) in TAK tissue samples. This approach can reveal spatial relationships between these pathways in disease contexts. Furthermore, serum or plasma from TAK patients can be analyzed for autoantibodies against MLX or its binding partners, potentially revealing novel biomarkers. Finally, for translational applications, high-throughput screening assays utilizing MLX antibodies could identify compounds that modulate the enhanced MLX-MondoA interaction associated with the Q139R variant, potentially leading to targeted therapeutic approaches for TAK.

How can researchers apply MLX antibodies to investigate the relationship between autophagy and inflammasome activation?

Researchers can strategically deploy MLX antibodies to dissect the complex relationship between autophagy and inflammasome activation, particularly given MLX's newly discovered role in both pathways . Begin with multiplex immunofluorescence using MLX antibodies combined with markers for autophagy (LC3, p62) and inflammasome components (NLRP3, ASC, caspase-1) to visualize their spatial relationships and co-localization patterns in cells expressing either wild-type MLX or the Q139R variant. This approach can reveal how MLX mutations affect the physical interaction between these pathways.

Implement co-immunoprecipitation with MLX antibodies followed by western blotting for autophagy regulators (like mTOR, LARS1) and inflammasome components to identify direct or indirect protein interactions that mediate cross-talk between these pathways . Since the Q139R mutation inhibits autophagy in lipopolysaccharide-stimulated cells while enhancing inflammasome activation , use MLX antibodies in time-course experiments to track the sequence of molecular events following inflammatory stimuli.

For functional studies, combine MLX antibody-based approaches with live-cell imaging of autophagy (using LC3-GFP) and inflammasome activation (using fluorescent caspase-1 substrates) in cells expressing different MLX variants. This can reveal the temporal relationship between autophagy inhibition and inflammasome activation. Extend these investigations to in vivo models by using MLX antibodies for immunohistochemistry in tissues from models of inflammatory diseases, correlating MLX expression patterns with markers of autophagy and inflammasome activation.

For mechanistic insights, use MLX antibodies in ChIP-seq experiments to identify transcriptional targets that may mediate the cross-regulation of autophagy and inflammasome pathways. Complement this with RNA-seq of cells expressing different MLX variants to create comprehensive pathway maps. Finally, develop high-content screening assays incorporating MLX antibodies to identify compounds that can selectively modulate the autophagy-inflammasome relationship, potentially leading to therapeutic approaches for inflammatory diseases like Takayasu arteritis where the MLX Q139R variant plays a pathogenic role .