PLIN5 Antibody

What is PLIN5 and why is it important for researchers studying lipid metabolism?

PLIN5 (Perilipin 5) is a lipid droplet-associated protein that plays a crucial role in coupling intramyocellular triacylglycerol lipolysis with fatty acid metabolism. It is particularly important because it helps regulate lipid storage and oxidation in tissues with high metabolic activity.

PLIN5 functions include:

• Regulation of lipid droplet formation and stability

• Coupling lipid droplet lipolysis with metabolic demand for fatty acids

• Recruiting mitochondria to lipid droplet surfaces through its C-terminal region (specifically amino acids 343-463)

• Protection against oxidative damage and lipotoxicity by controlling local fatty acid flux

PLIN5 is particularly relevant for researchers studying metabolic diseases, cardiovascular function, and lipid metabolism in oxidative tissues .

What are the various names and designations for PLIN5 antibody targets in literature?

When searching for PLIN5 antibodies, researchers should be aware of the various nomenclature used in scientific literature:

Understanding these alternative designations is essential when conducting literature searches and comparing antibody specificities across studies .

Which tissues typically express high levels of PLIN5?

PLIN5 shows a tissue-specific expression pattern primarily in metabolically active tissues:

• High expression: Heart, oxidative skeletal muscle (soleus, red quadriceps), brown adipose tissue, liver

• Moderate expression: Mixed skeletal muscle

• Lower expression: White adipose tissue, white glycolytic muscle

• Also detected in: Pancreatic β-cells

This tissue distribution correlates with PLIN5's role in tissues with high fatty acid oxidation capacity. When selecting positive control tissues for antibody validation, oxidative tissues like heart and red skeletal muscle are recommended .

What are the critical epitopes for PLIN5 antibody recognition and how do they affect experimental outcomes?

Several commercially available PLIN5 antibodies target different epitopes, with the C-terminal region being particularly important:

• The C-terminal region (amino acids 451-463) contains a highly immunogenic sequence (CPVKHTLMPELDF) that is targeted by many antibodies

• The PLIN5 antibody from Progen (#GP31) targets amino acids 451-463 at the C-terminus

• Other antibodies target regions between amino acids 305-453

The choice of epitope can significantly impact experimental outcomes:

• C-terminal antibodies may not detect truncated PLIN5 variants

• Some antibodies may have reduced reactivity when PLIN5 is phosphorylated

• C-terminal targeting is particularly relevant as this region is responsible for mitochondrial interaction

For critical applications, using multiple antibodies targeting different epitopes is recommended to validate findings .

How can researchers effectively validate PLIN5 antibody specificity in their experimental systems?

Proper validation of PLIN5 antibodies is crucial for reliable results. A comprehensive validation approach includes:

• Positive and negative tissue controls:

  • Use heart or oxidative muscle tissues as positive controls

  • Use tissues from PLIN5 knockout mice as negative controls

  • Compare expression in oxidative versus glycolytic muscles (expected higher expression in oxidative muscles)

• Molecular validation approaches:

  • Western blot should show a band at the expected molecular weight (50-55 kDa)

  • Validate with recombinant PLIN5 protein as a positive control

  • Perform siRNA or CRISPR knockout of PLIN5 and confirm loss of signal

• Cross-reactivity assessment:

  • Test for cross-reactivity with other perilipin family members

  • Use concentration-matched serum from the host species as a negative control

• Subcellular localization verification:

  • PLIN5 should localize to lipid droplets and partially to mitochondria

  • Co-staining with lipid droplet dyes (e.g., Oil Red O) should show colocalization .

What are the optimal conditions for immunoprecipitation of PLIN5?

For successful immunoprecipitation (IP) of PLIN5, researchers should consider the following protocol elements:

• Antibody selection:

  • Guinea pig anti-PLIN5 antibody (Progen Biotechnik, Heidelberg, Germany) has been successfully used for IP

  • Use antibodies validated for IP applications specifically

• Buffer composition:

  • IP buffer should be compatible with lipid droplet-associated proteins

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylated PLIN5

• Sample preparation:

  • For tissues with lipid droplets, special homogenization techniques may be required

  • For liver samples, homogenize in lysis buffer containing protease inhibitors

  • Incubate on ice with periodic vortexing (e.g., every 5 minutes for 15 minutes)

• IP procedure:

  • Follow manufacturer's instructions for immunoprecipitation kits (e.g., Pierce's Classic Immunoprecipitation Kit)

  • Use 20-40 μg/lane of protein for subsequent Western blot analysis

• Controls:

  • Include IgG control from the same species as the PLIN5 antibody

  • Consider using PLIN5 knockout samples as negative controls .

How does phosphorylation affect PLIN5 function and detection by antibodies?

PLIN5 phosphorylation is a critical regulatory mechanism that affects its function and potentially antibody recognition:

• Key phosphorylation sites:

  • S155 is the primary PKA phosphorylation site

  • S161 and S163 are additional potential phosphorylation sites

  • These sites are highly conserved across vertebrates

• Functional consequences of phosphorylation:

  • Phosphorylation of S155 is required for control of lipid metabolism

  • In basal conditions, PLIN5 inhibits lipid droplet hydrolysis

  • When phosphorylated (during stimulated conditions), this inhibition is lifted, allowing fatty acid release for β-oxidation

• Impact on antibody detection:

  • Phosphorylation may alter epitope accessibility or recognition

  • Phospho-specific antibodies can be used to monitor PLIN5 activation status

  • Consider using phosphatase treatment of samples if total PLIN5 detection is required

• Experimental approaches to study phosphorylation:

  • In vitro phosphorylation using PKA and [γ-32P] ATP

  • Mass spectrometry analysis of phosphopeptides

  • Use of phosphorylation-defective mutants (e.g., S155A) .

What is the role of PLIN5 in recruiting mitochondria to lipid droplets and how can this be studied?

PLIN5 uniquely mediates the physical association between lipid droplets and mitochondria, which is critical for coordinated lipid metabolism:

• Mechanistic basis:

  • PLIN5's C-terminal region (amino acids 343-463) is necessary for mitochondrial recruitment

  • PLIN5 expression increases contact points between lipid droplets and mitochondria

• Functional consequences:

  • Facilitates efficient transfer of fatty acids from lipid droplets to mitochondria for β-oxidation

  • Helps regulate fatty acid flux to protect mitochondria from lipotoxicity

  • Coordinates lipolysis with mitochondrial fatty acid oxidation capacity

• Experimental approaches to study this interaction:

  • Fluorescence microscopy: Using fluorescently tagged PLIN5 constructs and mitochondrial markers

  • Electron microscopy: For ultrastructural analysis of lipid droplet-mitochondria contacts

  • Proximity ligation assays: To detect close associations between PLIN5 and mitochondrial proteins

  • Immunogold labeling: For transmission electron microscopy to visualize PLIN5 localization

• Critical controls:

  • Comparison with other perilipin family members that do not recruit mitochondria

  • Use of deletion constructs lacking the C-terminal mitochondrial recruitment domain

  • Analysis of PLIN5 knockout/knockdown cells to confirm specific effects .

What are the optimal fixation and staining methods for PLIN5 immunohistochemistry?

Successful immunohistochemical detection of PLIN5 requires careful attention to tissue preparation and staining protocols:

• Fixation methods:

  • For paraffin-embedded sections: 4% paraformaldehyde fixation is commonly used

  • For frozen sections: Fixation in 4% paraformaldehyde solution with 0.1% Triton X-100 for 30 minutes

• Antigen retrieval:

  • For paraffin sections: TE buffer pH 9.0 is suggested as the primary method

  • Alternative: Citrate buffer pH 6.0 may also be effective

• Blocking conditions:

  • Block in PBS with 1% BSA for 1 hour at room temperature

  • Use concentration-matched normal serum from the antibody host species as negative control

• Antibody conditions:

  • Primary antibody dilutions: 1:50-1:500 (antibody-dependent)

  • Overnight incubation at 4°C is typically recommended

  • For PLIN5/lipid droplet co-staining: Combine with Oil Red O staining

• Visualization methods:

  • For brightfield: HRP-conjugated secondary antibodies with DAB substrate

  • For fluorescence: Fluorophore-conjugated secondary antibodies

  • Counter-staining: Hematoxylin for nuclear visualization in brightfield

  • For mitochondrial co-staining: Use antibodies against oxidative phosphorylation complexes I-V (Total OXPHOS) .

How can researchers distinguish between PLIN5 and other perilipin family members in their experiments?

Distinguishing PLIN5 from other perilipin family members requires careful experimental design:

• Antibody selection strategies:

  • Use antibodies targeting unique regions not conserved among perilipin family members

  • Validate antibody specificity using recombinant proteins of all perilipin family members

  • Consider using antibodies raised against synthetic peptides specific to PLIN5

• Expression pattern analysis:

  • PLIN5 is predominantly expressed in oxidative tissues (heart, red muscle)

  • PLIN1 is predominantly expressed in adipose tissue

  • PLIN2 has broader tissue distribution

  • These tissue-specific patterns can help confirm identity

• Molecular weight differentiation:

  • PLIN5: 51-55 kDa

  • PLIN1: ~60 kDa

  • PLIN2: ~50 kDa

  • PLIN3: ~47 kDa

  • PLIN4: ~140 kDa

• Functional assays:

  • Only PLIN5 recruits mitochondria to lipid droplets

  • Different perilipins have distinct effects on lipid droplet dynamics and lipolysis regulation

  • Use functional readouts characteristic of PLIN5 to confirm identity .

What are the most effective methods to study PLIN5 deletion effects in experimental models?

Various approaches can be used to study the effects of PLIN5 deletion or knockdown:

• Genetic models:

  • Plin5-/- knockout mice show decreased triacylglycerol content in heart (52% reduction)

  • Tissue-specific differences: reduced triacylglycerol in red quadriceps but increased in white quadriceps

  • β-galactosidase can be used as a reporter for PLIN5 deletion in certain knockout models

• In vitro manipulation approaches:

  • siRNA knockdown in cell culture models

  • CRISPR/Cas9-mediated knockout in cell lines

  • In vivo DNA electrotransfer for muscle-specific overexpression or knockdown

• Readouts to assess PLIN5 deletion effects:

  • Lipid droplet content and morphology (Oil Red O staining)

  • Triacylglycerol quantification

  • Mitochondrial function (Seahorse XF analyzer)

  • Fatty acid metabolism (radiometric methodology)

  • Oxidative stress markers (TBARS, MDA levels, SOD activity)

  • Cellular lipid composition analysis

• Control considerations:

  • Use littermate controls for genetic models

  • For in vivo electroporation, use contralateral limb as internal control

  • Include rescue experiments by re-expressing PLIN5 to confirm specificity of observed phenotypes .

How should researchers approach contradictory findings in PLIN5 antibody-based experiments?

When faced with contradictory findings in PLIN5 research, consider these methodological approaches:

• Antibody validation issues:

  • Test multiple antibodies targeting different epitopes

  • Confirm specificity using PLIN5 knockout/knockdown samples

  • Consider the impact of post-translational modifications on epitope recognition

• Tissue and context specificity:

  • PLIN5 functions may differ between tissues (e.g., heart vs. liver vs. skeletal muscle)

  • Metabolic state affects PLIN5 expression and function

  • Oxidative vs. glycolytic muscle types show different PLIN5 expression patterns

• Technical factors:

  • Protein extraction methods can affect recovery of lipid droplet-associated proteins

  • Fixation protocols impact epitope preservation and accessibility

  • Antibody concentration and incubation conditions impact signal-to-noise ratio

• Experimental design considerations:

  • Use multiple detection methods (WB, IF, IHC) to corroborate findings

  • Include appropriate positive and negative controls

  • Consider using orthogonal approaches (e.g., mass spectrometry) to validate antibody-based findings

  • Examine PLIN5 in different physiological conditions (fasted vs. fed, exercised vs. sedentary) .

What are the recommended protocols for detecting PLIN5 phosphorylation in experimental samples?

Detecting PLIN5 phosphorylation requires specialized techniques:

• Mass spectrometry-based approaches:

  • Digest proteins with trypsin

  • Analyze by high-resolution mass spectrometry

  • Look for phosphopeptides containing S155, S161, and S163

  • Compare phosphopeptide abundance in control vs. stimulated conditions

• Radioactive labeling:

  • Incubate recombinant PLIN5 or cellular samples with PKA and [γ-32P] ATP

  • Monitor incorporation of radioactive phosphate by phosphorimaging

  • Compare wild-type PLIN5 with phosphorylation-defective mutants (S155A, S161A, S163A)

• Phospho-specific antibodies:

  • Use antibodies that specifically recognize phosphorylated S155

  • Compare signals before and after phosphatase treatment

  • Include phosphorylation-defective mutants as negative controls

• Functional readouts:

  • Monitor changes in lipid droplet hydrolysis under basal vs. PKA-stimulated conditions

  • Measure fatty acid release and oxidation rates

  • Compare effects of wild-type PLIN5 vs. phosphorylation-defective mutants

• In vivo phosphorylation detection:

  • Use PKA activators (e.g., β-adrenergic agonists) to stimulate PLIN5 phosphorylation

  • Perform immunoprecipitation followed by phospho-specific detection

  • Consider phospho-proteomic approaches for unbiased detection .

How is PLIN5 antibody detection used to understand metabolic diseases?

PLIN5 antibody detection provides valuable insights into metabolic disease mechanisms:

• Oxidative stress and lipotoxicity:

  • PLIN5 protects against oxidative damage in multiple cell types

  • In pancreatic β-cells, PLIN5 abrogates lipotoxic stress through enhanced antioxidant defense

  • PLIN5 upregulation decreases reactive oxygen species production

  • Enhances cellular glutathione levels and antioxidant enzyme expression

• Cardiovascular disease:

  • PLIN5 provides cardioprotection against ischemia/reperfusion injury

  • Deficiency exacerbates myocardial infarct area and ventricular dysfunction

  • PLIN5 decreases free fatty acid peroxidation by inhibiting lipid droplet lipolysis

  • PLIN5 interacts with SERCA2 and promotes calcium handling in cardiac tissue

• Cancer research:

  • PLIN5 is downregulated in ovarian cancer tissues through hypermethylation

  • Demethylated PLIN5 can suppress tumor growth, cell proliferation, migration, and invasion

  • PLIN5 methylation status can potentially serve as a biomarker in cancer

• Metabolic disorders:

  • PLIN5 regulates lipid storage and oxidation balance

  • Its dysregulation contributes to lipid accumulation disorders

  • PLIN5 phosphorylation state affects metabolic flexibility .

What are the current limitations of PLIN5 antibodies and how can researchers address them?

Current PLIN5 antibodies have several limitations that researchers should be aware of:

• Cross-reactivity issues:

  • Some antibodies may cross-react with other perilipin family members

  • Solution: Validate using PLIN5 knockout samples and multiple antibodies targeting different epitopes

• Post-translational modification interference:

  • Phosphorylation may affect antibody binding to certain epitopes

  • Solution: Use phosphatase treatment or multiple antibodies targeting different regions

• Species specificity limitations:

  • Not all antibodies work across multiple species

  • Solution: Carefully check reactivity information and validate in your specific species

• Detection method constraints:

  • Some antibodies work well for Western blot but poorly for immunohistochemistry

  • Solution: Select antibodies validated for your specific application

• Quantification challenges:

  • Lipid droplet-associated proteins can be difficult to extract completely

  • Solution: Use standardized extraction protocols specifically designed for lipid droplet proteins

• Batch-to-batch variability:

  • Especially with polyclonal antibodies

  • Solution: Test each new lot against a reference sample and consider monoclonal alternatives .

How can PLIN5 antibody detection be combined with other techniques for comprehensive lipid metabolism studies?

Integrating PLIN5 antibody detection with complementary techniques provides deeper insights:

• Co-immunoprecipitation studies:

  • Use PLIN5 antibodies to pull down protein complexes

  • Identify interaction partners through mass spectrometry

  • Study how these interactions change under different metabolic conditions

• Live cell imaging approaches:

  • Combine with fluorescently-tagged lipid droplet dyes

  • Monitor dynamics of PLIN5-positive lipid droplets

  • Assess mitochondrial-lipid droplet interactions in real time

• Metabolic flux analysis:

  • Correlate PLIN5 expression/localization with fatty acid oxidation rates

  • Use radiometric methodologies to track fatty acid metabolism

  • Employ Seahorse XF analyzer to measure mitochondrial function

• Multi-omics integration:

  • Combine PLIN5 protein data with:

    • Lipidomics to assess lipid species composition

    • Transcriptomics to identify coordinated gene expression patterns

    • Metabolomics to measure metabolic intermediates

• In vivo functional assessment:

  • Correlate tissue PLIN5 levels with physiological parameters

  • Use tissue-specific overexpression or knockdown

  • Monitor whole-body metabolism in PLIN5 genetic models .

What are the emerging roles of PLIN5 beyond canonical lipid droplet regulation?

Recent research has uncovered unexpected roles for PLIN5 beyond its classical function:

• Mitochondrial quality control:

  • PLIN5 may influence mitochondrial morphology and function

  • PLIN5-deficient myocardium exhibits severely damaged mitochondria

  • May play a role in mitochondrial turnover and biogenesis

• Antioxidant defense pathways:

  • PLIN5 induces the Nrf2-ARE system, a master regulator of cellular adaptive response to oxidative stress

  • Activates PI3K/Akt and ERK signaling pathways

  • Enhances expression of antioxidant enzymes like glutamate-cysteine ligase and heme oxygenase-1

• Calcium handling:

  • Interacts with SERCA2 in cardiac tissue

  • Promotes calcium handling and cardiac contractility

  • May influence excitation-contraction coupling

• Tumor suppression:

  • Acts as a tumor suppressor in ovarian cancer

  • Methylation status affects its expression in cancer cells

  • Inhibits cell proliferation, migration, and invasion

• Transcriptional regulation:

  • May influence gene expression patterns

  • Enrichment analysis identified upregulated gene ontology terms related to contraction in high vs. low PLIN5 expression .

How is PLIN5 antibody research contributing to potential therapeutic approaches?

PLIN5 research is revealing potential therapeutic avenues for several diseases:

• Cardioprotective strategies:

  • PLIN5 protects against ischemia/reperfusion injury

  • Targeting PLIN5 expression or activity could provide cardioprotection

  • Understanding PLIN5 phosphorylation could lead to interventions that modulate its protective effects

• Metabolic disorder treatments:

  • Modulating PLIN5 activity could help balance lipid storage and utilization

  • Potential target for obesity, diabetes, and fatty liver disease

  • PLIN5's role in β-cells suggests relevance for diabetes therapies

• Cancer therapeutics:

  • Epigenetic drugs to reverse PLIN5 hypermethylation in cancers

  • 5-Aza-dC (DNA methyltransferase inhibitor) can restore PLIN5 expression

  • Potential biomarker for cancer diagnosis or prognosis

• Antioxidant strategies:

  • PLIN5's role in enhancing antioxidant defense could inform treatments for oxidative stress-related conditions

  • Understanding the PI3K/Akt and ERK pathways activated by PLIN5 could reveal new therapeutic targets

• Exercise mimetics:

  • PLIN5 is involved in metabolic adaptations to exercise

  • Targeting its activity could potentially mimic some beneficial effects of exercise

  • Relevant for conditions where exercise capacity is limited .