Phospho-LIPE (Ser552) Antibody

What is Phospho-LIPE (Ser552) Antibody and what is its target?

Phospho-LIPE (Ser552) Antibody specifically detects endogenous levels of hormone-sensitive lipase (HSL) protein only when phosphorylated at the Serine 552 residue. This antibody recognizes the phosphorylated form of HSL within the amino acid region 518-567 of the human HSL protein. The antibody is generated by immunizing rabbits with synthesized peptides derived from this region, containing the phosphorylated Ser552 site. It serves as an important tool for studying HSL activation and regulation in lipolysis research, as phosphorylation at Ser552 represents one of the key regulatory modifications of this enzyme .

What applications can Phospho-LIPE (Ser552) Antibody be used for?

Phospho-LIPE (Ser552) Antibody has been validated for multiple experimental applications, making it versatile for various research approaches:

These applications enable researchers to examine HSL phosphorylation status across different experimental contexts, from protein expression levels to subcellular localization .

What species reactivity has been confirmed for this antibody?

The Phospho-LIPE (Ser552) Antibody has been experimentally validated to react with samples from:

• Human

• Mouse

• Rat

This cross-species reactivity is based on the high conservation of the Ser552 phosphorylation site and surrounding amino acid sequence across these species. The antibody was generated against a human HSL peptide sequence, but due to sequence homology in this region, it successfully recognizes the equivalent phosphorylated sites in mouse and rat HSL proteins .

How should samples be prepared to preserve phosphorylation status for detection?

Preserving phosphorylation status is critical for accurate Phospho-LIPE (Ser552) detection. Follow these methodological guidelines:

• Tissue/Cell Harvest: Rapidly harvest and flash-freeze samples in liquid nitrogen to prevent phosphatase activity.

• Lysis Buffer Composition: Use a phosphatase-preserving lysis buffer containing:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 1 mM EDTA

  • Critical components:

    • Phosphatase inhibitors (10 mM sodium fluoride, 2 mM sodium orthovanadate, 10 mM β-glycerophosphate)

    • Protease inhibitor cocktail

• Temperature Control: Maintain samples at 4°C throughout processing and avoid repeated freeze-thaw cycles.

• Denaturing Conditions: Add SDS sample buffer immediately after lysis and heat samples at 95°C for 5 minutes to inactivate phosphatases.

• Gel Electrophoresis Considerations: Use freshly prepared SDS-PAGE gels with appropriate percentage (8-10% recommended) to resolve the 116 kDa HSL protein .

What are the optimal blocking and antibody incubation conditions for Western blotting?

To achieve optimal signal-to-noise ratio when detecting phosphorylated HSL (Ser552):

• Membrane Transfer: Transfer proteins to PVDF membrane (preferred over nitrocellulose for phospho-epitopes).

• Blocking Solution:

  • 5% BSA (not milk) in TBS-T (TBS with 0.1% Tween-20) for 1 hour at room temperature

  • BSA is critical as milk contains phosphoproteins that can interfere with phospho-antibody detection

• Primary Antibody Incubation:

  • Dilute Phospho-LIPE (Ser552) Antibody at 1:1000 in 5% BSA/TBS-T

  • Incubate overnight at 4°C with gentle rocking

  • For stronger signals, extend incubation up to 24 hours

• Washing Protocol: Wash 4-5 times with TBS-T, 5-10 minutes each

• Secondary Antibody Incubation:

  • Anti-rabbit HRP-conjugated at 1:5000 in 5% BSA/TBS-T

  • Incubate for 1 hour at room temperature

• Detection System: ECL substrates optimized for phospho-protein detection .

What validation methods confirm Phospho-LIPE (Ser552) Antibody specificity?

Robust validation is essential to ensure experimental results reflect true phosphorylation events:

• Peptide Competition Assay: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides:

  • Signal abolishment with phospho-peptide but not with non-phospho-peptide confirms specificity

  • Evidence shows complete signal blocking when using the specific phospho-peptide in both Western blot and IHC applications

• Phosphatase Treatment Control:

  • Treat half of your sample with lambda phosphatase

  • Loss of signal in treated samples confirms phospho-specificity

• Stimulation/Inhibition Controls:

  • Stimulate cells with agents known to induce HSL phosphorylation (e.g., isoproterenol)

  • Treat cells with AMPK activators to assess impact on HSL phosphorylation

  • Signal enhancement/reduction upon treatment validates specificity

• Knockdown/Knockout Validation:

  • Compare signal in LIPE knockout cells/tissues versus wild-type

  • Absence of signal in knockout samples confirms target specificity

How does phosphorylation at Ser552 regulate HSL function compared to other phosphorylation sites?

HSL regulation involves multiple phosphorylation sites with distinct functional implications:

• Ser552 Phosphorylation (Human numbering):

  • Increases intrinsic enzyme activity by ~2-fold

  • Mediated primarily by Protein Kinase A (PKA) following β-adrenergic stimulation

  • Creates conformational changes that improve substrate access to the active site

• Comparative Phosphorylation Effects:

  • Ser649 and Ser650: Primary activation sites, increasing activity 3-5 fold

  • Ser554: Secondary PKA site, moderate activity enhancement

  • Ser565: AMPK-mediated phosphorylation, inhibits HSL activity

  • Ser552: Enhances activity and may facilitate translocation to lipid droplets

• Functional Relevance:

  • While Ser552 phosphorylation contributes to activation, phosphorylation by AMPK can reduce HSL translocation to lipid droplets, indicating a complex regulatory mechanism

  • Sequential phosphorylation at multiple sites likely required for maximal HSL activation

Researchers should consider monitoring multiple phosphorylation sites simultaneously for comprehensive understanding of HSL regulation.

What experimental designs can assess HSL phosphorylation dynamics in metabolic studies?

To investigate temporal and stimulus-dependent HSL phosphorylation:

• Time-Course Experiments:

  • Stimulate adipocytes/tissues with lipolytic agents (isoproterenol, forskolin)

  • Harvest samples at multiple timepoints (0, 5, 15, 30, 60, 120 minutes)

  • Analyze phosphorylation at Ser552 via Western blot with quantitative densitometry

  • Compare with other phosphorylation sites using site-specific antibodies

• Metabolic Challenge Protocols:

  • Fasting/Refeeding: Examine HSL phosphorylation in adipose tissue during:

    • Fed state (baseline)

    • Short-term fasting (4-6 hours)

    • Extended fasting (12-24 hours)

    • Refeeding after fasting

  • Exercise Intervention: Compare pre-exercise, immediate post-exercise, and recovery periods

  • Cold Exposure: Monitor HSL phosphorylation during adaptive thermogenesis

• Spatial Distribution Analysis:

  • Combine immunofluorescence with confocal microscopy to track:

    • Phospho-HSL (Ser552) translocation to lipid droplets

    • Co-localization with perilipin and other droplet-associated proteins

    • Quantitative image analysis of phospho-HSL distribution

What methodological approaches help resolve conflicting phospho-HSL (Ser552) data?

When facing discrepancies in phospho-HSL detection:

• Sample Preparation Variations:

  • Compare flash-frozen versus chemical fixation methods

  • Evaluate different protein extraction techniques

  • Assess influence of phosphatase inhibitor cocktail composition

• Antibody Validation:

  • Test multiple antibody clones/sources against the same samples

  • Perform simultaneous detection with total HSL antibody on split samples

  • Utilize antibody dilution series to identify optimal concentrations

• Normalization Strategies:

  • Normalize phospho-signal to total HSL protein rather than housekeeping proteins

  • Consider running phospho-HSL alongside total HSL on separate blots from the same samples

  • Implement multiple technical and biological replicates with statistical analysis

• Complementary Techniques:

  • Verify phosphorylation status using mass spectrometry

  • Employ phospho-specific ELISA for quantitative measurement

  • Consider in vitro kinase assays to confirm phosphorylation events

What are common problems encountered with Phospho-LIPE (Ser552) Antibody and their solutions?

Researchers frequently encounter these challenges when working with phospho-specific antibodies:

• High Background Signal:

  • Cause: Insufficient blocking or cross-reactivity

  • Solution: Increase BSA concentration to 5-10%, extend blocking time to 2 hours, add 0.1% Tween-20 to antibody dilution

• Weak or Absent Signal:

  • Cause: Phosphorylation degradation during sample handling

  • Solution: Ensure immediate addition of phosphatase inhibitors, maintain cold temperature throughout, avoid sample storage

• Multiple Bands:

  • Cause: Antibody cross-reactivity or protein degradation

  • Solution: Increase antibody dilution, reduce exposure time, prepare fresh samples with protease inhibitors

• Inconsistent Results Between Experiments:

  • Cause: Variation in phosphorylation status due to sample handling

  • Solution: Standardize sample collection protocol, process all comparative samples simultaneously, include positive controls

How should researchers optimize antibody dilution for different applications?

Systematic optimization is critical for each application:

• Western Blotting Optimization:

  • Start with manufacturer's recommended range (1:500-1:2000)

  • Prepare dilution series (e.g., 1:500, 1:1000, 1:2000, 1:4000)

  • Process identical samples at each dilution

  • Select dilution that provides optimal signal-to-noise ratio

  • Validated optimal dilution: 1:1000 for most tissue lysates

• Immunohistochemistry Optimization:

  • Begin with 1:100 dilution

  • Test serial dilutions on positive control tissues

  • Include negative controls (primary antibody omission, non-phosphorylated tissues)

  • Optimal dilution typically falls between 1:100-1:300 for paraffin sections

• Immunofluorescence Considerations:

  • Lower dilutions often required (1:50-1:200)

  • Test fixation methods (paraformaldehyde vs. methanol)

  • Optimize permeabilization conditions

  • Include phosphatase inhibitors in fixation buffers

How does long-term storage affect Phospho-LIPE (Ser552) Antibody performance?

Proper storage is crucial for maintaining antibody reactivity and specificity:

• Storage Recommendations:

  • Store at -20°C for up to one year (primary recommendation)

  • For frequent use, aliquot and store working portions at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles (more than 3-5 cycles significantly reduce activity)

• Degradation Indicators:

  • Progressive signal weakening across experiments

  • Increased background staining

  • Appearance of non-specific bands

• Stability Enhancement Strategies:

  • Add carrier protein (0.1% BSA) to antibody solution

  • Store in small aliquots (10-20 μL) to minimize freeze-thaw events

  • Include cryopreservatives like glycerol (final concentration 50%)

  • Monitor antibody performance with consistent positive controls over time

How can Phospho-LIPE (Ser552) Antibody be used to study lipolysis regulation in different metabolic conditions?

Phospho-LIPE (Ser552) Antibody enables detailed investigation of lipolysis regulation:

• Metabolic State Comparisons:

  • Analyze phosphorylation levels across metabolic conditions:

    • Fasting vs. fed states

    • Exercise vs. sedentary conditions

    • Insulin-stimulated vs. insulin-resistant states

  • Correlate phosphorylation levels with lipolytic rate measurements

• Hormonal Response Assessment:

  • Monitor Ser552 phosphorylation following exposure to:

    • Catecholamines (epinephrine, norepinephrine)

    • Glucagon

    • Insulin (inhibitory effect)

    • Tumor necrosis factor-alpha (TNFα)

  • Establish time-course and dose-response relationships

• Pathophysiological Models:

  • Compare HSL phosphorylation patterns in:

    • Diet-induced obesity

    • Genetic models of obesity/diabetes

    • Aging-related metabolic changes

    • Inflammatory conditions

  • Correlate findings with metabolic parameters (glucose tolerance, insulin sensitivity)

What methodological approaches can quantify changes in HSL phosphorylation at Ser552?

For precise quantification of phosphorylation changes:

• Densitometric Analysis:

  • Normalize phospho-HSL signal to total HSL protein

  • Use digital image acquisition and analysis software

  • Apply background subtraction algorithms

  • Generate phospho/total HSL ratios across conditions

• Phospho-Specific ELISA:

  • Develop sandwich ELISA using:

    • Capture antibody: total HSL

    • Detection antibody: phospho-HSL (Ser552)

  • Create standard curves with recombinant phosphorylated protein

  • Achieve higher throughput and quantitative precision

• Multiplexed Phosphorylation Analysis:

  • Employ multiplexed Western blotting to simultaneously detect:

    • Multiple HSL phosphorylation sites

    • Related signaling proteins (PKA, AMPK, perilipin)

  • Use fluorescently-labeled secondary antibodies with different wavelengths

  • Analyze signal co-localization and relative intensities

How can researchers correlate HSL phosphorylation with functional lipolytic activity?

Establishing the functional significance of phosphorylation requires:

• Parallel Activity Measurements:

  • Measure glycerol/fatty acid release from tissues/cells

  • Determine lipase enzyme activity using fluorescent substrates

  • Correlate with phosphorylation status at Ser552 and other sites

• Phosphorylation Site Mutations:

  • Generate Ser552Ala (phospho-null) mutations

  • Create Ser552Asp (phospho-mimetic) mutations

  • Compare enzymatic activity and lipid droplet association

  • Assess impact on lipolytic response to stimulation

• Pharmacological Interventions:

  • Apply kinase inhibitors (H89 for PKA, Compound C for AMPK)

  • Use phosphatase inhibitors (okadaic acid, calyculin A)

  • Monitor changes in both phosphorylation and lipolytic activity

  • Establish causative relationships between phosphorylation and function