mylipb Antibody

What is MYLIP and why is it important in research?

MYLIP (Myosin-regulated Light chain Interacting Protein) is a significant protein involved in multiple cellular processes. It functions primarily as an E3 ubiquitin ligase and plays crucial roles in lipid metabolism and cholesterol homeostasis by regulating the degradation of lipid-modifying enzymes. MYLIP is also known by several alternative names, including IDOL (Inducible Degrader of the LDL receptor), MIR, and E3 ubiquitin-protein ligase MYLIP. Recent research has identified MYLIP as a potential tumor suppressor gene in lung cancer, as its expression levels are positively correlated with better prognosis in lung cancer patients . The protein has a molecular weight of approximately 49.9 kDa and is localized in the cell membrane and cytoplasm as a peripheral membrane protein .

What are the validated experimental applications for MYLIP antibodies?

MYLIP antibodies have been validated for several research applications, with Western blotting (WB) and ELISA being the most commonly employed techniques . These antibodies enable researchers to detect and analyze MYLIP expression in various cell types and tissues, making them valuable tools for studying metabolism, cancer biology, and related fields. When selecting a MYLIP antibody, researchers should verify that it has been validated for their specific application of interest and is reactive to their species of study (human, mouse, rat, etc.) .

What positive control samples are recommended for validating MYLIP antibody specificity?

According to antibody product information, Jurkat cells (a human T lymphocyte cell line) and mouse spleen tissue are recommended as positive control samples for validating MYLIP antibody specificity . These samples are known to express detectable levels of MYLIP and can serve as reliable controls for antibody validation experiments. When establishing a new experimental system, researchers should include these positive controls alongside experimental samples to confirm antibody performance.

What is the recommended protocol for optimizing Western blot detection of MYLIP?

For optimal Western blot detection of MYLIP, researchers should follow this methodological approach:

• Sample preparation: Extract proteins using lysis buffer containing protease inhibitors and incubate at 4°C for 30 minutes.

• Centrifugation: Centrifuge samples at 12,000 rpm for 15 minutes at 4°C and collect the supernatant.

• Protein quantification: Determine protein concentration using the BCA method and standardize all samples.

• Sample loading: Load approximately 20 μg of protein per lane for electrophoresis.

• Protein transfer: Transfer proteins to PVDF membranes using standard transfer conditions.

• Blocking: Block membranes with 5% skim milk at room temperature for 2 hours.

• Primary antibody: Dilute MYLIP antibody at 1:500 to 1:1000 and incubate at 4°C overnight .

• Washing: Wash membranes 3 times with TBST buffer for 6 minutes per wash.

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.

• Detection: Visualize using chemiluminescent detection reagents.

• Analysis: Analyze band intensity using Image J or similar software .

How should researchers design experiments to investigate MYLIP's role in cancer cell biology?

Based on published research methodologies, a comprehensive experimental design would include:

• Expression analysis:

  • Compare MYLIP mRNA and protein expression between cancer and normal tissues/cells using RT-qPCR and Western blotting .

  • Analyze bioinformatics databases to correlate MYLIP expression with patient outcomes.

• Functional studies:

  • Generate stable MYLIP-overexpressing cancer cell lines using lentiviral vectors .

  • Verify overexpression by both RT-qPCR and Western blot.

• Proliferation assays:

  • Perform CCK8 assays at multiple timepoints (0, 24, 48, 72, and 96 hours).

  • Conduct colony formation experiments to assess long-term proliferation effects .

• Migration and invasion studies:

  • Implement scratch test assays with appropriate controls and timepoints.

  • Conduct transwell invasion assays using Matrigel-coated chambers .

• In vivo validation:

  • Establish xenograft models in nude mice using cells with modified MYLIP expression.

  • Monitor tumor growth systematically and measure final tumor volume and weight .

• Statistical analysis:

  • Apply appropriate statistical tests to determine significance of observed differences.

What are the critical factors to consider when performing real-time PCR for MYLIP mRNA quantification?

When quantifying MYLIP mRNA using real-time PCR, researchers should consider these methodological factors:

• RNA extraction quality:

  • Extract RNA using standardized protocols to ensure high quality.

  • Verify RNA integrity and purity through spectrophotometric analysis.

• Primer design and validation:

  • Use validated primers specific for MYLIP (e.g., forward: 5'-CCGCTGCACATCGTC-3' and reverse: 5'-CGTGGAAGGCGGTGATCAG-3') .

  • Verify primer specificity through melt curve analysis.

• PCR conditions optimization:

  • Implement appropriate cycling conditions (pre-denaturation at 94°C for 3 minutes, followed by 40 cycles of denaturation at 94°C for 4 seconds, annealing at 56°C for 5 seconds, and extension at 72°C for 6 seconds) .

• Reference gene selection:

  • Use GAPDH or other appropriate housekeeping genes as internal references.

  • Validate the stability of reference genes in your experimental system.

• Quantification method:

  • Calculate relative expression using the 2^(-ΔΔCt) method .

  • Include appropriate controls for normalization.

• Technical replicates:

  • Perform experiments in triplicate to ensure reproducibility.

  • Address outliers through appropriate statistical methods.

How can researchers effectively investigate MYLIP's E3 ubiquitin ligase activity in relation to its tumor suppressor function?

To investigate the relationship between MYLIP's E3 ubiquitin ligase activity and its tumor suppressor function, researchers should consider the following approaches:

• Structure-function analysis:

  • Generate MYLIP mutants with disrupted RING domain (responsible for E3 ligase activity).

  • Compare the effects of wild-type versus ligase-dead MYLIP on cancer cell phenotypes.

• Substrate identification:

  • Conduct co-immunoprecipitation experiments using MYLIP antibodies to pull down potential substrate proteins.

  • Perform mass spectrometry analysis to identify novel MYLIP-interacting proteins.

• Ubiquitination assays:

  • Assess the ubiquitination status of putative substrate proteins in the presence or absence of MYLIP.

  • Compare ubiquitination patterns between wild-type and mutant MYLIP.

• Degradation kinetics:

  • Measure the half-life of substrate proteins in cells with varying levels of MYLIP expression.

  • Use cycloheximide chase assays to track protein stability over time.

• Signaling pathway analysis:

  • Investigate how MYLIP-mediated ubiquitination affects key oncogenic signaling pathways.

  • Examine downstream effectors using phospho-specific antibodies and pathway inhibitors.

What techniques can be employed to resolve discrepancies between MYLIP mRNA and protein expression data?

When facing discrepancies between MYLIP mRNA and protein levels, researchers should consider these methodological approaches:

• Technical validation:

  • Verify primer specificity for mRNA detection using sequencing.

  • Confirm antibody specificity using appropriate controls.

  • Use multiple antibodies targeting different MYLIP epitopes.

• Biological mechanisms investigation:

  • Post-transcriptional regulation: Assess microRNA targeting of MYLIP mRNA.

  • mRNA stability: Measure MYLIP mRNA half-life using actinomycin D chase experiments.

  • Translation efficiency: Perform polysome profiling to assess MYLIP mRNA translation.

  • Protein stability: Conduct cycloheximide chase experiments to determine MYLIP protein half-life.

• Temporal analysis:

  • Track both mRNA and protein levels over time to identify temporal relationships.

  • Consider time-course experiments following perturbations to the system.

• Cell-type specificity:

  • Evaluate whether discrepancies are cell-type dependent.

  • Compare expression patterns across multiple cell lines.

• Integrated analysis:

  • Use mathematical modeling to understand relationships between transcription, translation, and degradation rates.

  • Consider systems biology approaches to place MYLIP in broader regulatory networks.

How can researchers effectively design in vivo experiments to validate MYLIP's tumor suppressor role?

For robust in vivo validation of MYLIP's tumor suppressor function, researchers should implement the following experimental design elements:

• Animal model selection:

  • Use immunocompromised mice (e.g., 4-week-old male nude mice) to establish xenograft models .

  • Consider genetically engineered mouse models for studying MYLIP in specific cancer types.

• Experimental groups:

  • Include multiple experimental groups to compare:

    • Normal lung cells (negative control)

    • Cancer cells with endogenous MYLIP expression

    • Cancer cells with MYLIP overexpression

    • If possible, cancer cells with MYLIP knockdown/knockout

• Cell preparation and injection:

  • Prepare standardized cell suspensions (e.g., 5×10^6 cells) .

  • Use consistent injection techniques and sites.

• Measurement parameters:

  • Monitor tumor growth systematically at regular intervals (e.g., every 3 days) .

  • Measure both tumor volume and weight as endpoints.

  • Consider survival analysis where appropriate.

• Molecular analysis of tumors:

  • Perform immunohistochemistry to verify MYLIP expression in tumors.

  • Analyze proliferation markers (Ki-67, PCNA) and apoptosis markers.

  • Examine angiogenesis and tumor microenvironment.

• Statistical considerations:

  • Ensure adequate sample sizes based on power analysis.

  • Apply appropriate statistical tests for data analysis.

  • Control for multiple comparisons.

What are common sources of variability in MYLIP Western blotting, and how can they be addressed?

Common sources of variability in MYLIP Western blotting include:

• Sample preparation issues:

  • Problem: Inconsistent protein extraction or degradation.

  • Solution: Standardize lysis conditions; use fresh protease inhibitors; maintain samples at 4°C during processing .

• Antibody performance:

  • Problem: Batch-to-batch variability or loss of activity.

  • Solution: Validate each new antibody lot; store antibodies according to manufacturer recommendations; avoid freeze-thaw cycles.

• Detection sensitivity:

  • Problem: Weak signals for endogenous MYLIP.

  • Solution: Optimize antibody dilution (recommended 1:500 to 1:1000) ; increase protein loading; use enhanced chemiluminescence systems.

• Non-specific binding:

  • Problem: Multiple bands or high background.

  • Solution: Optimize blocking conditions; increase washing steps; adjust antibody concentration; include appropriate controls.

• Inconsistent loading:

  • Problem: Uneven protein loading affects band intensity.

  • Solution: Carefully quantify protein; use reliable loading controls; normalize to housekeeping proteins.

• Membrane transfer issues:

  • Problem: Inefficient protein transfer.

  • Solution: Optimize transfer conditions; verify transfer efficiency with reversible staining.

• Data analysis variability:

  • Problem: Subjective interpretation of band intensity.

  • Solution: Use digital imaging systems with appropriate exposure settings; implement quantitative analysis software.

How should researchers approach optimization of immunohistochemistry protocols for MYLIP detection in tissue samples?

When optimizing immunohistochemistry protocols for MYLIP detection:

• Tissue processing considerations:

  • Optimize fixation time to prevent epitope masking.

  • Use standardized tissue processing protocols.

  • Consider tissue-specific modifications based on protein abundance.

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic).

  • Optimize pH, temperature, and duration of antigen retrieval.

  • Consider high-pressure antigen retrieval for difficult samples.

• Antibody protocol refinement:

  • Titrate antibody concentration to find optimal signal-to-noise ratio.

  • Test different incubation times and temperatures.

  • Consider using antibody diluents with signal enhancers.

• Detection system selection:

  • Choose appropriate detection systems based on expected MYLIP expression levels.

  • Consider polymer-based detection systems for higher sensitivity.

  • Evaluate chromogenic versus fluorescent detection based on research needs.

• Controls implementation:

  • Include tissues with known MYLIP expression as positive controls (e.g., Jurkat cells, mouse spleen) .

  • Use MYLIP-negative tissues or antibody omission as negative controls.

  • Consider MYLIP-overexpressing tissues as additional positive controls.

• Quantification strategy:

  • Develop consistent scoring methods for MYLIP staining.

  • Consider digital pathology approaches for objective quantification.

  • Validate scoring through multiple independent observers.

What experimental strategies can researchers use to investigate MYLIP's interaction with the LDL receptor and other potential binding partners?

To study MYLIP's interactions with the LDL receptor and other binding partners:

• Co-immunoprecipitation (Co-IP):

  • Use MYLIP antibodies to pull down MYLIP complexes from cell lysates.

  • Perform Western blotting to detect co-precipitated binding partners.

  • Include appropriate controls to validate specificity of interactions.

• Proximity ligation assay (PLA):

  • Utilize MYLIP antibodies together with antibodies against potential interactors.

  • This technique allows visualization of protein-protein interactions in situ with high specificity.

• Immunofluorescence co-localization:

  • Perform dual immunofluorescence labeling with MYLIP antibodies and antibodies against potential partners.

  • Analyze co-localization using confocal microscopy and quantitative co-localization analysis.

• Protein domain mapping:

  • Generate MYLIP truncation or deletion mutants to identify interaction domains.

  • Use pull-down assays with recombinant protein fragments.

  • Validate findings using full-length proteins in cellular contexts.

• Functional interaction studies:

  • Assess how MYLIP affects the stability and localization of putative binding partners.

  • Examine how overexpression or knockdown of MYLIP impacts partner protein function.

  • Investigate reciprocal effects of partner proteins on MYLIP activity.

How can MYLIP antibodies be utilized to explore the relationship between lipid metabolism and cancer progression?

MYLIP antibodies can be instrumental in exploring the lipid metabolism-cancer connection through:

• Expression correlation studies:

  • Analyze MYLIP expression in relation to key lipid metabolism markers in cancer tissues.

  • Perform immunohistochemistry on tissue microarrays containing samples from patients with detailed lipid profiles.

• Metabolic pathway intersection:

  • Investigate how MYLIP-mediated regulation of LDLR impacts cholesterol uptake in cancer cells.

  • Examine relationships between MYLIP expression and fatty acid synthesis pathways.

• Therapeutic targeting approaches:

  • Screen for compounds that modulate MYLIP expression or activity.

  • Assess how lipid-lowering drugs affect MYLIP expression in cancer models.

• Microenvironment studies:

  • Explore how tumor-associated macrophages and adipocytes influence MYLIP expression in cancer cells.

  • Examine MYLIP expression in hypoxic versus normoxic tumor regions.

• Clinical correlation analysis:

  • Correlate MYLIP expression with patients' lipid profiles and metabolic parameters.

  • Investigate whether MYLIP expression predicts response to metabolic interventions.

What methodological approaches can be used to study the differential expression of MYLIP across normal and pathological lung tissues?

To study differential MYLIP expression across lung tissues:

• Multi-level expression analysis:

  • Perform RT-qPCR for mRNA quantification from patient-matched normal and cancer tissues .

  • Use Western blotting with MYLIP antibodies for protein-level confirmation .

  • Implement immunohistochemistry for spatial distribution analysis.

• Single-cell analysis approaches:

  • Apply single-cell RNA sequencing to identify cell type-specific MYLIP expression patterns.

  • Use single-cell proteomics to correlate MYLIP protein with other markers.

  • Implement multiplexed immunofluorescence to map MYLIP expression in the tissue microenvironment.

• Comprehensive tissue profiling:

  • Analyze MYLIP expression across different lung cancer subtypes.

  • Compare expression in primary tumors versus metastatic sites.

  • Examine expression changes during disease progression.

• Technical considerations:

  • Use laser capture microdissection to isolate specific tissue regions.

  • Implement tissue microarrays for high-throughput analysis.

  • Consider spatial transcriptomics for mapping expression patterns.

• Validation approaches:

  • Corroborate findings across multiple patient cohorts.

  • Validate with animal models recapitulating human disease.

  • Perform functional studies in relevant cell types.

How can researchers develop integrated experimental approaches to comprehensively characterize MYLIP's tumor suppressor functions?

An integrated approach to characterizing MYLIP's tumor suppressor functions should include:

• Multi-omics profiling:

  • Transcriptomics: RNA-seq of cells with modulated MYLIP expression.

  • Proteomics: Mass spectrometry to identify MYLIP-dependent proteome changes.

  • Metabolomics: Analysis of lipid profiles and metabolic changes.

  • Epigenomics: Investigation of chromatin modifications associated with MYLIP expression.

• Mechanistic investigations:

  • Identify direct MYLIP substrates in cancer cells.

  • Characterize signaling pathways affected by MYLIP expression.

  • Determine how MYLIP affects cell cycle regulation and apoptosis.

• Comprehensive phenotypic analysis:

  • Beyond proliferation, migration, and invasion, examine:

    • Cell differentiation status

    • Response to therapeutic agents

    • Metabolic reprogramming

    • Immune interactions

• Advanced in vivo models:

  • Patient-derived xenografts with modulated MYLIP expression.

  • Genetically engineered mouse models.

  • Orthotopic models that recapitulate tumor microenvironment.

• Clinical correlation:

  • Analyze MYLIP expression in relation to comprehensive patient data.

  • Investigate associations with response to specific therapies.

  • Explore potential as a biomarker for patient stratification.

• Therapeutic implications:

  • Develop strategies to restore MYLIP expression or activity in cancers with low expression.

  • Investigate combination approaches targeting both MYLIP and related pathways.