mylipa Antibody

What is MYLIP and why is it important to study?

MYLIP (myosin regulatory light chain interacting protein), also known as Idol (inducible degrader of the LDL receptor), is a critical E3 ubiquitin ligase that regulates cholesterol metabolism by facilitating the degradation of LDL receptors. MYLIP belongs to the ERM protein family, whose members function as cytoskeletal effector proteins linking actin to membrane-bound proteins at the cell surface . The importance of MYLIP stems from its role in cholesterol homeostasis, as it mediates the ubiquitination of LDL receptor, leading to enhanced receptor degradation, likely via the lysosomes .

The LXR-MYLIP/Idol pathway has been demonstrated to reduce LDL-R levels in a tissue-specific manner, influencing plasma cholesterol levels . This mechanism represents a novel level of LDL-R regulation via protein ubiquitination that may facilitate the development of new strategies for treating high blood cholesterol in various diseases.

What applications are MYLIP antibodies suitable for in research?

MYLIP antibodies have been validated for multiple research applications, with different products showing specific performance characteristics:

MYLIP antibodies have demonstrated reactivity with human, mouse, rat, and hamster samples, making them versatile tools for comparative studies across species .

How should I choose between monoclonal and polyclonal MYLIP antibodies?

The choice between monoclonal and polyclonal MYLIP antibodies depends on your specific research objectives:

Monoclonal antibodies (e.g., Boster Bio A05031, clone 5G12G2):

• Provide high specificity to a single epitope (for MYLIP, often within amino acids 161-210)

• Offer consistent performance between batches

• Ideal for applications requiring reproducibility over time

• Better for detecting specific conformational changes or post-translational modifications

Polyclonal antibodies (e.g., AMSBIO AMS.AP5315c):

• Recognize multiple epitopes on the MYLIP protein

• Provide stronger signal by binding to several sites

• More tolerant to minor protein denaturation or modifications

• Better for detection of proteins in various sample preparation conditions

For detecting MYLIP in novel experimental conditions or in specific species not previously tested, polyclonal antibodies might provide more reliable detection initially. Once specific conditions are established, monoclonal antibodies can offer more consistent results for longitudinal studies .

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

For optimal Western blot results with MYLIP antibodies, follow these methodological steps:

• Sample preparation: Prepare tissue (human cerebellum) or cell lysates (e.g., CHO, HeLa cells) in RIPA buffer with protease inhibitors .

• Protein loading: Load 20-50 μg of total protein per lane.

• Electrophoresis and transfer: Use standard SDS-PAGE followed by transfer to PVDF or nitrocellulose membrane.

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute MYLIP antibody 1:500-1:1000 (for Proteintech 15455-1-AP) or 1:500-1:2000 (for Boster Bio A05031) in blocking buffer. Incubate overnight at 4°C .

• Washing: Wash 3-5 times with TBST, 5 minutes each.

• Secondary antibody: Apply appropriate secondary antibody (anti-rabbit IgG) at manufacturer's recommended dilution. Incubate for 1-2 hours at room temperature.

• Development: Use ECL or similar detection system.

Expected results: MYLIP should be detected at 46-50 kDa (Proteintech reports) or 39 kDa (Boster Bio) . This difference in observed molecular weight may be due to different sample types or gel conditions, and researchers should validate the specific band in their experimental system.

How do I optimize immunohistochemistry protocols for MYLIP detection in tissue samples?

For successful IHC detection of MYLIP in tissue samples:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissues. MYLIP has been successfully detected in human testis, brain, heart, kidney, liver, lung, ovary, placenta, skin, and spleen tissues .

• Antigen retrieval: Use TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 as alternative .

• Blocking: Block with 10% normal serum from the same species as secondary antibody for 1 hour.

• Primary antibody: Apply MYLIP antibody at 1:20-1:200 dilution (for Proteintech 15455-1-AP). Incubate overnight at 4°C .

• Washing and detection: Follow standard IHC protocols with appropriate detection system.

• Important considerations:

  • Titrate antibody concentration in each testing system to obtain optimal results

  • Include appropriate positive controls (e.g., human cerebellum tissue)

  • Include negative controls (primary antibody omitted or isotype control)

This protocol may require optimization depending on specific tissue type and fixation conditions.

How can I validate the specificity of my MYLIP antibody?

Validating MYLIP antibody specificity is crucial for ensuring reliable research results. Implement these methodological approaches:

• Blocking peptide experiment:

  • Pre-incubate the MYLIP antibody with its specific immunizing peptide (e.g., synthetic peptide derived from human MYLIP, AA range 161-210 for Boster Bio A05031)

  • Run parallel assays with blocked and unblocked antibody

  • Specific signals should diminish or disappear in the blocked antibody experiment

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes of MYLIP

  • Compare detection patterns across applications (WB, IHC, etc.)

  • Consistent patterns across antibodies increase confidence in specificity

• Genetic approaches:

  • Use MYLIP knockdown/knockout samples as negative controls

  • Overexpression systems as positive controls

  • These genetic validations provide the strongest evidence of specificity

• Cross-reactivity testing:

  • Test the antibody against closely related proteins

  • Evaluate reactivity across multiple species (human, mouse, rat samples)

Remember that even monoclonal antibodies can sometimes exhibit dual specificity or cross-reactivity with other antigens , necessitating thorough validation.

What are the challenges in detecting MYLIP in different sample types?

Researchers face several challenges when detecting MYLIP across different experimental samples:

• Varying expression levels: MYLIP expression is regulated by the LXR pathway and can vary significantly between tissues and under different conditions, affecting detection sensitivity requirements .

• Molecular weight variations: MYLIP has been reported at different molecular weights:

  • Calculated molecular weight: 50 kDa

  • Observed molecular weight: 46-50 kDa (Proteintech) or 39 kDa (Boster Bio)

  • These discrepancies may result from:

    • Post-translational modifications

    • Splice variants

    • Degradation products

    • Different electrophoresis conditions

• Sample-dependent optimization: As noted in technical documentation, MYLIP antibody performance is "sample-dependent" . Each experimental system requires titration of antibody dilution and optimization of protocol conditions.

• Cross-reactivity concerns: MYLIP shares structural features with other ERM family proteins, potentially leading to cross-reactivity . Thorough validation is required when studying tissues with high expression of related proteins.

To address these challenges, researchers should include appropriate positive controls (e.g., human cerebellum tissue, CHO cells) and validate their protocols extensively for each new experimental system.

How can I troubleshoot weak or non-specific signals when using MYLIP antibodies?

When encountering issues with MYLIP antibody performance, systematic troubleshooting approaches can help:

For weak signals:

• Antibody concentration: Try increasing antibody concentration gradually (e.g., from 1:1000 to 1:500 for WB) .

• Incubation conditions: Extend primary antibody incubation time (overnight at 4°C instead of 1-2 hours).

• Detection system: Use a more sensitive detection method (e.g., enhanced chemiluminescence plus).

• Sample preparation: Ensure samples are fresh and contain sufficient MYLIP protein. Consider enrichment techniques if expression is low.

• Antigen retrieval: For IHC, optimize antigen retrieval methods. Try both recommended methods: TE buffer pH 9.0 and citrate buffer pH 6.0 .

For non-specific signals:

• Blocking optimization: Increase blocking agent concentration or duration.

• Antibody dilution: Use more diluted antibody solution.

• Washing steps: Increase number and duration of washing steps.

• Secondary antibody: Ensure secondary antibody is appropriate for the host species (rabbit for most MYLIP antibodies) .

• Cross-reactivity testing: Perform blocking peptide experiments to identify and eliminate non-specific binding .

Remember that diligent optimization is required for each new experimental system, as stated in technical guidance: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

How do post-translational modifications affect MYLIP antibody detection?

MYLIP undergoes post-translational modifications that can influence antibody detection:

• Ubiquitination: As an E3 ubiquitin ligase, MYLIP not only ubiquitinates the LDL receptor but may also undergo auto-ubiquitination . This modification can:

  • Alter epitope accessibility

  • Change protein migration patterns in SDS-PAGE

  • Affect antibody binding affinity

• Phosphorylation: Potential phosphorylation sites exist within MYLIP that may be regulated by cellular signaling pathways. These modifications can:

  • Create or mask antibody binding sites

  • Influence protein-protein interactions

  • Affect functional activity

• Considerations for antibody selection:

  • Epitope location: Antibodies targeting regions prone to modification (like the RING domain involved in ubiquitination) may show variable results

  • Multiple antibodies: Using antibodies recognizing different regions of MYLIP can help distinguish modification-dependent effects

  • Phospho-specific antibodies: Consider whether phosphorylation status is relevant to your research question

For studies focusing on MYLIP regulation and function, researchers should consider how these modifications might impact detection and interpretation of results.

What are the appropriate controls for MYLIP antibody experiments?

Rigorous controls are essential for reliable interpretation of MYLIP antibody experiments:

Positive controls:

• Human cerebellum tissue (for WB and IHC)

• CHO cells (for WB)

• HeLa cells (for WB)

Negative controls:

• Primary antibody omission

• Isotype control antibody (using same species and isotype as MYLIP antibody)

• Blocking peptide competition (pre-incubation of antibody with immunizing peptide)

• MYLIP knockout/knockdown samples (gold standard)

Application-specific controls:

For Western blot:

• Loading control (β-actin, GAPDH)

• Molecular weight marker to confirm band size (expected 46-50 kDa or 39 kDa)

For Immunohistochemistry:

• Known positive tissue sections

• Serial sections with primary antibody omission

• Comparison with published MYLIP expression patterns

For quantitative applications:

• Standard curve using recombinant MYLIP protein

• Internal reference controls for normalization

How does MYLIP antibody performance compare across different species samples?

MYLIP antibody performance varies across species, reflecting both sequence conservation and technical considerations:

When working with species not listed in the validation data, consider:

• Sequence homology: Check epitope sequence conservation between your species of interest and validated species

• Cross-reactivity: Test antibody on known positive control from your species of interest

• Alternative antibodies: If available, use antibodies specifically raised against your species of interest

• Validation requirements: More extensive validation is required when using antibodies in non-validated species

What are the implications of MYLIP research for understanding diseases related to cholesterol metabolism?

MYLIP/Idol research using specific antibodies has significant implications for understanding cholesterol-related diseases:

• LDL receptor regulation: MYLIP/Idol is induced by LXR agonists in macrophages and liver, leading to ubiquitination of the LDL-R and enhanced receptor degradation . This represents a novel regulatory mechanism that could be therapeutically targeted.

• Cardiovascular disease: High plasma LDL cholesterol is a known risk factor for cardiovascular disease and atherosclerosis. MYLIP's role in regulating LDL-R levels makes it a potential target for new therapeutic strategies .

• Research approaches using MYLIP antibodies:

  • Monitoring MYLIP expression in response to pharmacological interventions

  • Studying tissue-specific regulation of the LXR-MYLIP pathway

  • Evaluating MYLIP protein interactions with LDL-R and other potential targets

  • Investigating MYLIP expression in patient samples with dysregulated cholesterol metabolism

• Therapeutic potential: Understanding MYLIP expression and regulation could lead to novel approaches for modulating LDL-R levels and cholesterol metabolism, potentially complementing current therapies like statins .

How can I optimize MYLIP antibody storage and handling for maximum performance?

Proper storage and handling of MYLIP antibodies are crucial for maintaining their performance over time:

Storage conditions:

• Store lyophilized antibodies at -20°C (stable for one year after shipment)

• After reconstitution, store at 4°C for short-term use (up to one month)

• For long-term storage of reconstituted antibodies, aliquot and store at -20°C

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

Reconstitution protocols:

• Briefly centrifuge the vial to ensure all material is at the bottom

• For lyophilized antibodies, reconstitute in double-distilled water or as recommended by manufacturer

• For Proteintech 15455-1-AP, no reconstitution needed (supplied in liquid form with PBS, 0.02% sodium azide, and 50% glycerol, pH 7.3)

• For Boster Bio A05031, supplied in liquid form with PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide

Working solution preparation:

• Prepare fresh dilutions on the day of experiment

• Use high-quality, sterile buffers for dilution

• Include preservatives (0.02% sodium azide) for solutions stored more than 24 hours

• Return stock antibody to recommended storage conditions immediately after use

Following these guidelines will help maintain antibody performance and extend its usable lifespan.

What advanced research applications employ MYLIP antibodies beyond standard detection methods?

Beyond standard detection applications, MYLIP antibodies are valuable tools for advanced research techniques:

• Co-immunoprecipitation studies:

  • Investigating MYLIP interactions with LDL receptor and other potential substrates

  • Identifying novel protein interaction partners

  • Studying protein complex formation under different cellular conditions

• ChIP-seq analysis:

  • When combined with DNA analysis, can help identify genomic regions affected by MYLIP-mediated regulation

  • Useful for studying global effects of MYLIP pathway activation

• Live cell imaging:

  • Using fluorescently-tagged MYLIP antibodies in permeabilized cells

  • Studying dynamic changes in MYLIP localization

  • Visualizing protein trafficking events

• Therapeutic development:

  • Screening compounds that modulate MYLIP-LDL receptor interactions

  • Evaluating effects of potential drugs on MYLIP expression and function

  • Characterizing MYLIP in patient samples to identify disease-specific patterns

• Multi-omics integration:

  • Correlating MYLIP protein levels (detected by antibodies) with transcriptomic data

  • Integrating proteomics and metabolomics data to understand MYLIP's role in cholesterol metabolism networks

These advanced applications extend the utility of MYLIP antibodies beyond simple protein detection to address complex biological questions related to lipid metabolism and cardiovascular disease.

How can I leverage immunosignature approaches with MYLIP antibodies for broader research insights?

Immunosignature approaches, which involve binding antibodies to random peptide libraries, can be combined with MYLIP antibody research for enhanced insights:

• Epitope mapping applications:

  • Characterizing the precise binding sites of MYLIP antibodies

  • Identifying mimotopes that may bind as strongly or stronger than the original antigen

  • Assessing whether MYLIP antibodies recognize linear or conformational epitopes

• Cross-reactivity profiling:

  • Evaluating potential cross-reactivity with other proteins

  • Identifying unexpected binding patterns that might reveal new biological insights

  • Distinguishing between specific and polyspecific binding characteristics

• Methodological considerations:

  • Use microarrays of random peptides to assess antibody properties

  • Apply different peptide libraries with varying lengths and compositions

  • Analyze binding patterns to detect preferences for:

    • Shorter vs. longer peptides

    • N-terminal vs. C-terminal motifs

    • Specific amino acid sequences

• Data interpretation:

  • Antibodies with polyspecific binding may exhibit different functional properties than highly specific antibodies

  • Common sequence motifs from peptide binding can predict potential interactions

  • These patterns may reveal unexpected functional relationships for MYLIP