Mylip Antibody

What are the principal applications for MYLIP antibodies in research?

MYLIP antibodies are primarily utilized in Western Blot (WB), Immunohistochemistry (IHC), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunofluorescence (IF) applications. Western blotting represents the most validated application across commercial antibodies, with recommended dilutions typically ranging from 1:500 to 1:2000 . When designing experiments, researchers should select antibodies specifically validated for their intended application, as performance can vary significantly between techniques. For immunohistochemistry applications, dilutions of 1:50 to 1:200 are typically recommended .

How should MYLIP antibodies be stored to maintain optimal activity?

Most commercial MYLIP antibodies are supplied in PBS buffer containing preservatives such as sodium azide (0.02-0.09%) and stabilizers like glycerol (often 50%) . Optimal storage conditions generally require -20°C for long-term preservation . For working solutions, storage at 4°C is recommended, though repeated freeze-thaw cycles should be avoided as they can compromise antibody performance. High-titer antibodies may retain activity for approximately one week at 4°C, but researchers should validate performance if recycling antibodies for multiple experiments .

What is the expected molecular weight when detecting MYLIP?

The observed molecular weight of MYLIP protein is approximately 49.7 kDa, with a calculated molecular weight of 49.91 kDa . When performing Western blot analysis, researchers should anticipate bands around 50 kDa. Variation from this expected weight may indicate post-translational modifications, protein degradation, or antibody cross-reactivity. When validating a new MYLIP antibody, positive controls such as transfected HEK293T cells overexpressing MYLIP can provide confirmation of specificity .

Which biological samples show reliable MYLIP expression for experimental controls?

Research indicates that MYLIP is expressed at varying levels across tissues. Notably, lung cancer tissue samples show significantly lower MYLIP expression compared to adjacent normal tissues, both at mRNA and protein levels (P<0.05) . This differential expression pattern makes paired lung cancer/normal tissue samples valuable for validation studies. Bioinformatic analyses of existing databases can help identify tissues with high or low MYLIP expression for experimental design. Cell lines such as A549 and H460 with manipulated MYLIP expression levels have been used successfully in functional studies .

How can researchers differentiate between isoforms or post-translationally modified forms of MYLIP?

Discriminating between MYLIP variants requires careful antibody selection and experimental design. MYLIP undergoes ubiquitination as part of its E3 ligase function, which can affect its apparent molecular weight in SDS-PAGE analysis. Researchers investigating specific post-translational modifications should:

• Select antibodies raised against specific regions or modifications of interest

• Perform immunoprecipitation followed by mass spectrometry analysis

• Use phosphatase or deubiquitinase treatments to confirm modification status

• Employ 2D gel electrophoresis to separate variants based on both charge and size

Commercial antibodies targeting the central region of MYLIP (e.g., amino acids 127-385) have demonstrated reliable detection in Western blot applications , while antibodies against different epitopes may be required for studying specific modifications.

What methodological approaches best demonstrate MYLIP's functional role in LDL receptor degradation?

MYLIP/IDOL has been established as an E3 ubiquitin ligase that targets the LDL receptor for degradation, influencing cholesterol metabolism. To effectively study this pathway, researchers should employ:

• Ubiquitination assays: Detecting polyubiquitinated LDL receptor using co-immunoprecipitation with MYLIP and Western blotting

• LDL receptor turnover studies: Pulse-chase experiments with cycloheximide treatment to measure LDLR degradation rates when MYLIP is overexpressed or silenced

• LDL uptake assays: Functional studies measuring fluorescently-labeled LDL uptake in cells with modulated MYLIP expression

• Localization studies: Immunofluorescence co-localization of MYLIP and LDL receptor in cellular compartments

Research has demonstrated that overexpression of MYLIP by adenoviral vectors reduces LDL receptor abundance and LDL uptake, while RNA silencing of endogenous MYLIP increases LDL receptor levels and enhances LDL uptake .

How can researchers address the seemingly contradictory roles of MYLIP in cholesterol metabolism versus tumor suppression?

MYLIP appears to have dual functions: regulating cholesterol metabolism through LDL receptor degradation and functioning as a potential tumor suppressor in lung cancer. These seemingly disparate roles present interesting research challenges:

• Tissue-specific expression analysis: Compare MYLIP expression and function across different tissues using immunohistochemistry with validated antibodies

• Substrate specificity studies: Identify tissue-specific ubiquitination targets using immunoprecipitation coupled with mass spectrometry

• Signaling pathway analysis: Investigate how MYLIP interacts with different signaling networks in various cellular contexts

• Domain-specific functional studies: Generate constructs with mutations in specific MYLIP domains to dissect which regions are responsible for each function

Recent research demonstrates that in lung cancer patients, MYLIP mRNA and protein expression are significantly lower in cancer tissues compared to normal adjacent tissues, and high MYLIP expression correlates with better prognosis. Functional studies show that MYLIP overexpression inhibits proliferation, migration, and invasion of lung cancer cells both in vitro and in vivo .

What experimental controls are critical when evaluating MYLIP expression in patient samples?

When analyzing MYLIP expression in clinical specimens, researchers should implement the following controls:

• Tissue-matched normal controls: Adjacent normal tissue from the same patient provides the most relevant comparison

• Antibody validation: Confirm antibody specificity using positive controls (MYLIP-overexpressing cells) and negative controls (MYLIP-knockdown cells)

• Multiple detection methods: Correlate protein expression (by Western blot or IHC) with mRNA levels (by RT-PCR)

• Quantitative analysis: Use standardized scoring methods for immunohistochemistry or densitometry for Western blots

Research has shown significant differences in MYLIP expression between cancer and normal tissues using these approaches. For example, RT-PCR results showed significantly lower MYLIP mRNA levels in lung tumor tissues compared to adjacent normal tissues, and Western blot analyses in 20 patients demonstrated significantly lower MYLIP protein levels in cancer samples compared to corresponding adjacent tissues .

What are the optimal protocols for detecting low levels of MYLIP expression?

When investigating tissues or cell lines with potentially low MYLIP expression, researchers should consider these approaches:

• Signal amplification systems: Use high-sensitivity detection reagents such as enhanced chemiluminescence (ECL) substrates

• Enrichment techniques: Perform immunoprecipitation before Western blotting

• Optimized antibody concentrations: Titrate antibody dilutions to maximize signal-to-noise ratio

• Extended exposure times: Longer exposure for Western blots, being careful to avoid background issues

• RNA analysis: Complement protein studies with qPCR for MYLIP mRNA

Studies examining MYLIP expression in lung cancer utilized Western blot analyses with appropriate controls to detect the relatively low expression levels in cancer tissues compared to normal adjacent tissues .

How can genetic polymorphisms in MYLIP affect antibody binding and experimental results?

The MYLIP gene contains known polymorphisms that may influence protein expression, structure, and function. The rs3757354 SNP, for example, has been associated with serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and apolipoprotein AI (ApoAI) levels . Researchers should consider:

• Epitope location: Select antibodies targeting conserved regions unaffected by common polymorphisms

• Genotyping samples: When possible, determine MYLIP genotypes in experimental samples

• Population diversity: Include samples from diverse genetic backgrounds when studying MYLIP function

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm findings

A study of the Guangxi Bai Ku Yao and Han populations demonstrated that the frequency of the G allele of rs3757354 was 49.92% in Bai Ku Yao and 56.27% in Han populations, with significant associations between genotypes and serum lipid parameters .

What methodological improvements can enhance reproducibility in MYLIP antibody-based experiments?

To ensure consistent and reliable results with MYLIP antibodies, researchers should implement:

• Standardized protocols: Detailed documentation of all experimental conditions

• Batch controls: Include standard samples across different experimental runs

• Antibody validation: Verify specificity through knockout/knockdown controls

• Quantification standards: Include recombinant MYLIP protein standards of known concentration

• Multiple detection methods: Complement antibody-based detection with orthogonal approaches

Studies investigating MYLIP expression have employed multiple methodologies, including RT-PCR and Western blot analyses, to validate findings and ensure reproducibility .

How might MYLIP's dual function in lipid metabolism and cancer progression inform therapeutic strategies?

MYLIP's involvement in both LDL receptor degradation and tumor suppression presents intriguing therapeutic possibilities:

• Targeted MYLIP modulation: Inhibitors of MYLIP might increase LDL receptor levels and improve cholesterol clearance, but could potentially promote cancer progression in susceptible tissues

• Tissue-specific targeting: Developing approaches that modulate MYLIP function in a tissue-specific manner

• Pathway-specific interventions: Targeting downstream effectors rather than MYLIP itself

• Biomarker development: Using MYLIP expression levels as prognostic indicators in cancer

Research indicates that MYLIP has significant inhibitory effects on the proliferation, migration, and invasion of lung cancer cells, suggesting it may function as a tumor suppressor . Simultaneously, MYLIP's role in LDL receptor degradation makes it relevant to cholesterol metabolism and potentially atherosclerosis .

What experimental approaches best elucidate the mechanisms by which MYLIP inhibits cancer progression?

To investigate MYLIP's tumor suppressor function, researchers should consider:

• Transcriptional target identification: ChIP-seq or RNA-seq analyses following MYLIP modulation

• Protein interaction networks: Immunoprecipitation coupled with mass spectrometry to identify binding partners

• Signaling pathway analysis: Phosphoproteomic analysis to identify altered signaling cascades

• In vivo models: Conditional knockout or overexpression models to assess tissue-specific effects

Studies have demonstrated that MYLIP overexpression inhibits proliferation, migration, and invasion of lung cancer cells in vitro, and tumor formation in vivo using nude mice. Experimental approaches included cell counting kit-8 (CCK8) assays, cell cloning experiments, scratch tests, invasion experiments, and tumor formation experiments in nude mice .

How can researchers reconcile contradictory findings regarding MYLIP expression and function across different tissues and disease states?

When addressing seemingly inconsistent results regarding MYLIP, researchers should implement:

• Comprehensive tissue profiling: Systematic analysis of MYLIP expression across multiple tissues and disease states

• Context-dependent functional studies: Investigating MYLIP function in different cellular environments

• Isoform-specific analysis: Determining if different MYLIP variants predominate in different tissues

• Regulatory network mapping: Identifying tissue-specific regulators of MYLIP expression and function

Research has shown varying MYLIP expression patterns, with particularly low expression in lung cancer tissues compared to normal adjacent tissues , while its role in cholesterol metabolism appears relevant across multiple tissue types .