MYL5 Antibody

What is MYL5 and what are its basic molecular characteristics?

MYL5 (myosin, light chain 5, regulatory) is a regulatory light chain protein with a calculated and observed molecular weight of 20 kDa. It functions as part of the myosin complex and is involved in various cellular physiological processes. The protein is encoded by the MYL5 gene (Gene ID: 4636), with its sequence available under GenBank Accession Number BC040050 and UniProt ID Q02045. As a component of the myosin machinery, MYL5 participates in cellular functions that require molecular motor activity, making it relevant to both normal physiological processes and pathological conditions .

What tissues express MYL5 and how can researchers detect it?

MYL5 expression has been detected in multiple tissues across different species. Western blot analysis has confirmed MYL5 expression in mouse skeletal muscle tissue, human brain tissue, and rat skeletal muscle tissue. Additionally, immunohistochemistry has successfully detected MYL5 in human cervical cancer tissue and mouse kidney tissue. For optimal detection, researchers should employ antibodies with confirmed reactivity to human, mouse, and rat samples, such as the 14249-1-AP polyclonal antibody. This antibody can be used for Western Blot (WB), Immunofluorescence (IF), Immunohistochemistry (IHC), and ELISA applications with appropriate dilutions (WB: 1:500-1:2000; IHC: 1:50-1:500) .

What are the recommended protocols for using MYL5 antibody in Western Blot and IHC?

For Western Blot applications with MYL5 antibody, researchers should use a dilution range of 1:500-1:2000. The protocol requires careful sample preparation from tissues known to express MYL5, such as skeletal muscle or brain tissue. For Immunohistochemistry (IHC), a dilution range of 1:50-1:500 is recommended. Antigen retrieval is a critical step for successful IHC staining; using TE buffer at pH 9.0 is suggested, though citrate buffer at pH 6.0 can serve as an alternative. It's important to note that optimal dilutions may be sample-dependent, so researchers should titrate the antibody in their specific testing systems to achieve optimal results. Standardized protocols are available from antibody manufacturers and should be followed while making appropriate adjustments for specific experimental conditions .

What is the relationship between MYL5 expression and immune cell infiltration in the tumor microenvironment?

MYL5 expression demonstrates significant correlations with tumor-infiltrating immune cells (TIICs) in breast cancer. Comprehensive analysis using databases such as TIMER, TIMER2.0, and TISIDB reveals that MYL5 expression negatively correlates with infiltration of various immune cell types, including cancer-associated fibroblasts, B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Particularly notable is the negative correlation between MYL5 expression and macrophage infiltration, especially tumor-associated macrophages (TAMs). As TAMs typically drive tumor progression, invasion, and metastasis, the negative correlation suggests that MYL5 might improve prognosis by regulating TAM activity in the tumor microenvironment. Additionally, MYL5 expression negatively correlates with markers of T cell exhaustion (including PD-1, PD-L1, and CTLA4), indicating that higher MYL5 levels might enhance T cell-mediated anti-tumor responses, potentially explaining the better survival outcomes observed in patients with elevated MYL5 expression .

What are the optimal conditions for using MYL5 antibody in different experimental applications?

For Western Blot applications, researchers should use MYL5 antibody at dilutions of 1:500-1:2000. Protein extraction should be performed under conditions that preserve MYL5's native structure, typically using RIPA buffer with protease inhibitors. When performing IHC, dilutions of 1:50-1:500 are recommended, with antigen retrieval using TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0). For immunofluorescence studies, researchers should optimize fixation methods (4% paraformaldehyde is commonly effective) and permeabilization conditions. The antibody storage conditions are critical for maintaining reactivity—store at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3). The antibody remains stable for one year after shipment, and aliquoting is unnecessary for -20°C storage. Researchers should perform validation experiments using positive control tissues (mouse skeletal muscle, human brain tissue, rat skeletal muscle) and include appropriate negative controls by omitting the primary antibody or using tissues known not to express MYL5 .

What approaches should researchers use to investigate MYL5's role in immune infiltration mechanisms?

To thoroughly investigate MYL5's role in immune infiltration, researchers should employ a multi-dimensional approach. First, conduct co-expression analyses between MYL5 and immune cell markers (such as CD8 for T cells, CD68 for macrophages, CD11c for dendritic cells) using qRT-PCR and immunoblotting in both tumor and adjacent normal tissues. Second, perform immunohistochemistry or multiplex immunofluorescence to visualize the spatial relationship between MYL5-expressing cells and immune infiltrates within the tumor microenvironment. Third, utilize flow cytometry to quantify immune cell populations in tissues with varying MYL5 expression levels. Fourth, employ in vitro co-culture systems with immune cells and cancer cells where MYL5 is either overexpressed or knocked down to evaluate functional interactions. Fifth, conduct in vivo experiments using MYL5 knockout or overexpression models to assess changes in immune infiltration patterns. Finally, analyze cytokine/chemokine profiles in relation to MYL5 expression to identify potential mediators of the observed immune cell recruitment or exclusion effects .

What controls are essential when using MYL5 antibody for cancer tissue analysis?

When analyzing cancer tissues with MYL5 antibody, several controls are essential to ensure reliable and interpretable results. First, positive tissue controls should include mouse skeletal muscle tissue, human brain tissue, or rat skeletal muscle tissue, which have been validated for MYL5 expression. Second, negative tissue controls should use tissues known not to express MYL5 or employ isotype control antibodies to assess non-specific binding. Third, when analyzing cancer tissues, researchers should always include adjacent normal tissue samples from the same patient for direct comparison of expression levels. Fourth, technical controls such as antibody dilution series should be performed to determine optimal concentration for specific tissue types. Fifth, when studying prognostic significance, include samples from patients with well-documented clinical outcomes across different disease stages. Sixth, for immune infiltration studies, incorporate staining for established immune cell markers in serial sections to correlate with MYL5 expression patterns. Finally, validation using alternative detection methods (e.g., in situ hybridization for MYL5 mRNA or multiple antibodies targeting different epitopes) strengthens the reliability of findings regarding MYL5 expression in cancer tissues .

What are common technical challenges when detecting MYL5 in clinical samples and how can they be overcome?

Researchers face several technical challenges when detecting MYL5 in clinical samples. First, variable fixation times in formalin-fixed paraffin-embedded (FFPE) tissues can affect epitope accessibility; this can be addressed by optimizing antigen retrieval methods, with TE buffer (pH 9.0) recommended for MYL5 detection, though citrate buffer (pH 6.0) provides an alternative. Second, background staining or non-specific signals may occur; these can be minimized by careful blocking (using 3-5% BSA or serum), titrating antibody dilutions (1:50-1:500 for IHC, 1:500-1:2000 for WB), and including appropriate negative controls. Third, degradation of MYL5 in archived samples may reduce detection sensitivity; using freshly prepared tissues or adjusting extraction methods for degraded samples can help. Fourth, heterogeneous expression within tumors may lead to sampling bias; this necessitates analyzing multiple regions from each sample. Fifth, cross-reactivity with other myosin light chains is possible; validation with alternative antibodies or complementary techniques (e.g., mass spectrometry) can confirm specificity. Finally, variations in tissue processing between institutions may affect reproducibility; standardizing protocols and including reference samples can ensure consistent results across different laboratories .

How can researchers distinguish between correlation and causation when analyzing MYL5's relationship with immune infiltration?

Distinguishing correlation from causation in MYL5's relationship with immune infiltration requires rigorous experimental approaches beyond observational data. First, researchers should move beyond correlative studies by implementing gain-and-loss-of-function experiments, using MYL5 overexpression and knockdown/knockout models to determine if changes in MYL5 levels directly alter immune cell recruitment or function. Second, temporal analyses examining whether MYL5 expression changes precede alterations in immune infiltration can help establish causative relationships. Third, mechanistic studies investigating how MYL5 affects signaling pathways related to immune function (such as cytokine production or chemokine signaling) provide insights into potential causal mechanisms. Fourth, in vivo models with conditional or inducible MYL5 modulation allow for temporal control to establish causation sequences. Fifth, analysis of downstream molecular events using techniques like ChIP-seq or RNA-seq after MYL5 modulation can identify direct transcriptional effects relevant to immune function. Finally, direct interaction studies using co-immunoprecipitation or proximity ligation assays can determine if MYL5 physically interacts with immune regulatory proteins, providing evidence for direct mechanistic links rather than statistical associations .

How does MYL5 expression correlate with immune checkpoint molecules and what are the therapeutic implications?

Recent studies have revealed significant correlations between MYL5 expression and immune checkpoint molecules in breast cancer. Notably, MYL5 expression negatively correlates with markers of T cell exhaustion, including PDCD1 (PD-1), CD274 (PD-L1), and CTLA4. This negative correlation suggests that tumors with higher MYL5 expression may have lower expression of these inhibitory immune checkpoint molecules, potentially leading to more active anti-tumor immune responses. These findings have important therapeutic implications, particularly for immune checkpoint inhibitor (ICI) therapies that target PD-1/PD-L1 or CTLA4 pathways. Patients with different MYL5 expression levels may respond differently to ICI therapy—higher MYL5 expression might indicate tumors with naturally lower immune checkpoint activity, potentially affecting the benefit derived from checkpoint blockade. Researchers should investigate whether MYL5 expression could serve as a biomarker for predicting response to immunotherapy. Additionally, the relationship between MYL5 and immune checkpoints suggests possible combination therapeutic strategies targeting both MYL5-related pathways and immune checkpoint molecules to enhance anti-tumor immunity .

What molecular pathways are enriched in correlation with MYL5 expression in cancer?

Analysis using the LinkedOmics database has identified significant enrichment of specific molecular pathways associated with MYL5 expression in breast cancer. Genes co-expressed with MYL5 are predominantly enriched in ATP-related and metabolism-related pathways, which aligns with MYL5's known functions in cellular energetics as part of the myosin complex. This suggests that MYL5 may influence cancer progression through metabolic regulation, particularly energy metabolism pathways critical for tumor growth and survival. Beyond metabolism, MYL5 expression correlates with genes involved in immune signaling pathways, further supporting its role in modulating the tumor immune microenvironment. The enrichment analysis indicates that MYL5 may function at the intersection of cellular metabolism and immune regulation, two critical aspects of cancer biology. Understanding these molecular associations provides mechanistic insights into how MYL5 might influence cancer outcomes and offers potential avenues for therapeutic intervention targeting these pathways. Further experimental validation of these enriched pathways is necessary to confirm direct functional relationships and identify specific molecular targets for cancer therapy .

How should researchers design experiments to investigate MYL5's functional role in cancer progression?

Designing experiments to investigate MYL5's functional role in cancer requires a comprehensive approach spanning in vitro and in vivo models. First, establish stable cancer cell lines with MYL5 overexpression and knockdown/knockout using techniques like CRISPR-Cas9 or shRNA. Second, characterize these modified cell lines through proliferation assays, migration/invasion assays, apoptosis assessment, and cell cycle analysis to determine how MYL5 affects fundamental cancer hallmarks. Third, perform xenograft experiments using these modified cell lines in immunodeficient mice to assess tumor growth, metastasis, and response to therapies. Fourth, for immune-related studies, utilize immunocompetent syngeneic mouse models where both cancer cells and host immune system have matching genetic backgrounds. Fifth, conduct co-culture experiments with cancer cells and immune cells to investigate direct interactions. Sixth, employ organoid or 3D culture systems that better recapitulate the tumor microenvironment. Finally, validate findings in patient-derived xenografts or patient samples through correlative studies. Throughout these experiments, researchers should incorporate appropriate controls and use multiple cell lines representing different cancer subtypes to ensure robust, generalizable results .

What experimental approaches can identify the mechanistic link between MYL5 and immune cell recruitment in tumors?

To establish the mechanistic link between MYL5 and immune cell recruitment, researchers should implement a multifaceted experimental strategy. Begin with chemotaxis assays using conditioned media from MYL5-overexpressing or -depleted cancer cells to assess their ability to attract immune cells in transwell systems. Conduct cytokine/chemokine profiling using multiplex ELISA or cytokine arrays to identify secreted factors that might mediate immune cell recruitment. Perform chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing on MYL5-modified cells to identify transcriptional targets that may influence immune signaling. Use pathway inhibitors targeting candidates identified from omics analyses to validate their role in MYL5-mediated immune modulation. Implement in vivo models with fluorescently labeled immune cells to track recruitment to tumors with varying MYL5 expression in real-time using intravital microscopy. Analyze clinical samples for spatial correlations between MYL5 expression and immune infiltration patterns using multiplex immunohistochemistry. Finally, investigate potential direct interactions between MYL5 and immune signaling molecules through co-immunoprecipitation or proximity ligation assays. This comprehensive approach can elucidate whether MYL5 influences immune cell recruitment directly or through downstream signaling cascades .

What considerations are important when developing MYL5 as a prognostic biomarker for clinical application?

Developing MYL5 as a clinical prognostic biomarker requires addressing several key considerations. First, standardize detection methods for MYL5 expression, determining whether immunohistochemistry, qRT-PCR, or other technologies provide the most reliable and reproducible measurements across different laboratories. Second, establish clear cutoff values to distinguish "high" versus "low" MYL5 expression through robust statistical methods like receiver operating characteristic (ROC) curve analysis on large patient cohorts. Third, validate MYL5's prognostic value in prospective clinical trials rather than relying solely on retrospective analyses, ensuring its independent prognostic significance when controlling for established clinicopathological factors. Fourth, determine if MYL5's prognostic value varies across cancer subtypes, stages, or in response to specific treatments to define the precise clinical contexts where it offers valuable information. Fifth, assess MYL5's performance compared to existing biomarkers and whether it provides additive prognostic information within multivariate models. Sixth, investigate MYL5's potential as a predictive biomarker for specific therapies, particularly immunotherapies, given its correlation with immune infiltration. Finally, develop practical, cost-effective assays suitable for routine clinical implementation, considering factors like turnaround time, sample requirements, and integration into existing diagnostic workflows .

What criteria should researchers use when selecting an MYL5 antibody for specific experimental applications?

When selecting an MYL5 antibody, researchers should evaluate several critical criteria based on their specific experimental applications. First, confirm species reactivity matches your experimental model; some antibodies (like 14249-1-AP) show reactivity with human, mouse, and rat samples, making them versatile across different model systems. Second, verify the antibody has been validated for your specific application (WB, IHC, IF, or ELISA) with published evidence or manufacturer validation data. Third, consider antibody type—polyclonal antibodies often provide higher sensitivity but potentially lower specificity, while monoclonal antibodies offer higher specificity but might recognize fewer epitopes. Fourth, check the immunogen used to generate the antibody; those raised against full-length proteins or larger fragments may recognize multiple epitopes. Fifth, review published literature using the specific antibody catalog number to assess performance in similar experimental contexts. Sixth, examine the recommended dilutions and protocols for your application (WB: 1:500-1:2000; IHC: 1:50-1:500 for 14249-1-AP). Finally, confirm the antibody's species of origin (e.g., rabbit IgG) is compatible with your secondary detection systems and won't create conflicts with other antibodies in multiplex experiments .

What are the optimal storage and handling conditions for maintaining MYL5 antibody activity?

Maintaining MYL5 antibody activity requires proper storage and handling conditions throughout the research process. Store MYL5 antibodies at -20°C in their recommended buffer (PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 for 14249-1-AP). The antibody remains stable for one year after shipment under these conditions. Unlike some antibodies, aliquoting is unnecessary for -20°C storage of this formulation, which simplifies handling. When working with the antibody, minimize freeze-thaw cycles by quickly removing the needed amount and returning the stock to -20°C promptly. During experiments, keep the working dilution on ice and use within the same day whenever possible. If the antibody contains BSA (as in the 20μl size of 14249-1-AP which contains 0.1% BSA), be aware of potential interactions with other reagents. For long-term storage beyond manufacturer recommendations, consider adding additional stabilizing proteins or commercial antibody stabilizers. Always check for signs of deterioration such as precipitates or unusual coloration before use. If reduced activity is observed after storage, titrating the antibody again may be necessary to determine if higher concentrations are required for optimal signal detection .

How should researchers address contradictory findings regarding MYL5's role in different cancer types?

When confronting contradictory findings about MYL5's role across cancer types, researchers should implement a systematic approach to reconciliation. Begin by critically evaluating methodological differences between studies, including sample sizes, detection techniques, cutoff values for expression categories, and statistical methods. Stratify analyses by cancer subtypes, as MYL5 may have context-dependent functions—what appears contradictory might reflect biologically relevant variations across cancer subtypes with distinct molecular characteristics. Consider tumor heterogeneity and microenvironmental factors, as MYL5's impact may depend on the composition of surrounding stromal and immune cells. Examine potential confounding factors such as treatment history, patient demographics, and comorbidities that might explain divergent results. Conduct meta-analyses combining multiple datasets with careful consideration of study quality and consistency of outcome measures. Design experiments that directly test hypotheses explaining contradictions, such as whether MYL5 functions differently depending on specific genetic backgrounds or oncogenic drivers. Finally, consider that contradictions might reflect true biological complexity—MYL5 could have dual roles as both tumor-promoting and tumor-suppressing depending on cancer type, stage, or microenvironmental context .

How can researchers design experiments to resolve contradictions in the literature regarding MYL5's function?

To resolve contradictions about MYL5's function, researchers should design comprehensive experiments that directly address the core inconsistencies. First, conduct parallel studies in multiple cancer types using identical methodologies to determine if contradictions reflect true biological differences between cancer types rather than technical variations. Second, implement crossover experimental designs where multiple research groups analyze the same samples using their respective protocols to identify method-dependent discrepancies. Third, perform conditional experiments testing MYL5's function under different microenvironmental conditions (hypoxia, inflammation, nutrient availability) to determine if contradictions reflect context-dependent functions. Fourth, utilize genetic approaches to engineer isogenic cell lines differing only in MYL5 expression levels while controlling for genetic background, eliminating confounding variables that might explain contradictory findings. Fifth, conduct time-course experiments to explore whether MYL5's function changes during cancer progression, potentially explaining why studies at different disease stages yield conflicting results. Sixth, apply systems biology approaches combining transcriptomics, proteomics, and functional assays to build comprehensive models of MYL5's role in different contexts. Finally, design in vivo experiments with tissue-specific and inducible MYL5 modulation to evaluate stage-specific functions that might reconcile seemingly contradictory observations. These approaches collectively address both technical and biological sources of contradiction in a systematic manner .