PLIN5 Antibody, FITC conjugated

What is PLIN5 and what cellular functions does it perform?

PLIN5, also known as OXPAT, LSDP5, or PAT-1, is a lipid droplet (LD) coat protein that promotes association of lipid droplets with mitochondria. It is primarily expressed in tissues with high fat-oxidative capacity, such as heart, skeletal muscles, and brown adipose tissue . PLIN5 plays multiple crucial roles:

• Regulates lipid metabolism, particularly fatty acid oxidation in various tissues

• Protects against cellular oxidative stress by reducing ROS levels

• Enhances mitochondrial function and increases expression of mitochondrial function-related genes

• Promotes LD formation and mitochondria-LD contact

• Reduces apoptotic rates in cells under stress conditions

Research has shown that PLIN5 overexpression increases cellular triglyceride storage while simultaneously increasing fatty acid oxidation and inducing expression of mitochondrial enzymes involved in oxidative metabolism . This suggests PLIN5's involvement in both triglyceride storage and oxidative degradation of fatty acids released from lipid droplets.

How does PLIN5 localize within cells and how does this relate to its function?

PLIN5 exhibits a unique dual localization pattern that distinguishes it from other perilipin family members:

• Primarily localizes to the surface of lipid droplets

• Also found in mitochondria, unlike other perilipins like PLIN2

• In fat oxidative type I muscle fibers, PLIN5 displays a staining pattern similar to recognized mitochondrial proteins

The presence of PLIN5 in both locations enables it to direct fatty acids from lipid droplets to mitochondria for oxidation, making it particularly important in tissues with high energetic demands . This spatial arrangement facilitates the coordination between lipid storage and energy production.

What applications is PLIN5 Antibody, FITC conjugated suitable for?

PLIN5 Antibody with FITC conjugation is applicable for various research techniques depending on the specific antibody product:

The FITC-conjugated antibody is particularly valuable for direct visualization applications without requiring secondary antibodies, making it ideal for multi-color immunofluorescence protocols where minimizing cross-reactivity is important .

What methodological considerations are important when using PLIN5 Antibody, FITC conjugated for immunofluorescence?

When using PLIN5 Antibody with FITC conjugation for immunofluorescence studies, researchers should consider:

• Sample preparation:

  • For tissue sections: Use freshly frozen or properly fixed tissues

  • For paraffin-embedded samples: Microwave treatment is recommended for antigen retrieval

  • For cultured cells: Cells can be labeled using the modified Oil-red-O staining protocol for simultaneous visualization of neutral lipids and PLIN5

• Optimization steps:

  • Begin with manufacturer's recommended dilution and optimize based on signal-to-noise ratio

  • Include proper positive controls (tissues known to express PLIN5 such as heart, oxidative skeletal muscle)

  • Include negative controls (tissues with low PLIN5 expression or antibody omission)

• Co-staining considerations:

  • PLIN5 can be co-stained with mitochondrial markers (e.g., OXPHOS antibodies) to study mitochondria-LD interactions

  • When studying lipid droplets, combine with neutral lipid stains like BODIPY493/503

• Signal detection:

  • FITC conjugate has excitation/emission peaks at 499/515 nm

  • Recommended laser line: 488 nm

  • Avoid prolonged exposure to light to prevent photobleaching

Proper storage of the antibody (aliquoted at -20°C, avoiding freeze/thaw cycles and light exposure) is critical for maintaining reactivity and fluorescence intensity .

How can PLIN5 Antibody be used to investigate oxidative stress mechanisms?

PLIN5 plays a significant role in protecting against oxidative stress, making it a valuable target for investigating oxidative damage mechanisms:

• Experimental approaches to measure PLIN5-mediated ROS reduction:

  • Use DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) method to quantify cellular ROS levels in cells with manipulated PLIN5 expression

  • Apply DHE (dihydroethidium) method to detect superoxide (O₂⁻- ) levels

  • Measure mitochondrial membrane potential using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine) method

  • Assess cytochrome c release from mitochondria to cytoplasm via cellular fractionation and Western blotting

• Study design for oxidative stress experiments:

  • Generate cells with PLIN5 overexpression or knockdown

  • Induce oxidative stress with hydrogen peroxide (200 μM) or lipopolysaccharide (LPS)

  • Compare ROS levels, mitochondrial function, and cell survival between control and PLIN5-manipulated cells

  • Test protective effects by measuring apoptotic rates using flow cytometry

• Investigation of molecular pathways:

  • Examine the JNK-p38-ATF pathway activation in response to PLIN5 manipulation

  • Analyze expression of mitochondrial function-related genes (COX2, COX4, CS)

  • Investigate anti-oxidant gene expression (GPX1, GPX2, SOD1, SOD2, TXNRD1, CAT, PRDX3)

  • Study the Nrf2-ARE system activation through PI3K/Akt and ERK signal pathways

Research has demonstrated that PLIN5 overexpression decreases cellular ROS levels while PLIN5 knockdown increases ROS levels. This effect persists even when cells are challenged with oxidative stressors like hydrogen peroxide .

What approaches can be used to study PLIN5's role in lipid metabolism and mitochondrial function?

To investigate PLIN5's dual role in lipid storage and mitochondrial function:

• Lipid droplet analysis techniques:

  • Quantify lipid droplet number and size using BODIPY493/503 staining in cells with altered PLIN5 expression

  • Perform co-localization analysis of PLIN5 with lipid droplets and mitochondria using confocal microscopy

  • Use electron microscopy with immunogold labeling to visualize PLIN5 distribution at ultrastructural level

• Mitochondrial function assessment:

  • Measure fatty acid oxidation rates using ¹⁴C-palmitate oxidation assays in muscle homogenates

  • Compare oxidation rates between samples with normal and overexpressed PLIN5

  • Isolate mitochondria to determine direct effects of PLIN5 on mitochondrial function

  • Analyze expression of genes related to mitochondrial oxidative capacity (e.g., COX and CS)

• In vivo manipulation approaches:

  • Use in vivo DNA electrotransfer technique to achieve overexpression of PLIN5 in specific tissues

  • Compare control limb/tissue with PLIN5-overexpressing limb/tissue within the same animal

  • Analyze metabolic parameters, lipid content, and mitochondrial function

• Gene expression analysis:

  • Examine transcriptional changes in response to PLIN5 manipulation

  • Focus on genes involved in fatty acid metabolism, mitochondrial function, and antioxidant defense

  • Correlate changes in gene expression with functional outcomes

Research has demonstrated that PLIN5 overexpression increases complete fatty acid oxidation by 44.8% in muscle homogenates, supporting PLIN5's role in directing fatty acids from lipid droplets to mitochondrial oxidation .

What are common challenges when using PLIN5 Antibody, FITC conjugated and how can they be addressed?

Researchers working with PLIN5 Antibody, FITC conjugated may encounter several technical challenges:

• Weak or absent fluorescence signal:

  • Cause: Inadequate antibody concentration, improper storage, photobleaching

  • Solution: Optimize antibody dilution, store at -20°C in aliquots, minimize light exposure, ensure tissue expression of PLIN5

• High background fluorescence:

  • Cause: Non-specific binding, inadequate blocking, autofluorescence

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include autofluorescence quenching steps, use proper negative controls

• Signal specificity concerns:

  • Cause: Cross-reactivity with other perilipin family members

  • Solution: Validate antibody specificity using PLIN5 knockout/knockdown samples, compare staining pattern with published results

• Tissue-dependent variability:

  • Cause: Different expression levels across tissues, different fixation requirements

  • Solution: Adjust protocol based on tissue type, include positive controls (heart, oxidative muscle)

• Storage-related degradation:

  • Cause: Repeated freeze-thaw cycles, improper temperature, exposure to light

  • Solution: Store antibody in small aliquots at -20°C, avoid repeated freeze-thaw cycles, protect from light

For optimal results, researchers should validate the antibody in their specific experimental system before proceeding with critical experiments, particularly when studying tissues or cell types not previously characterized for PLIN5 expression.

How can researchers validate the specificity of PLIN5 Antibody?

Validating antibody specificity is crucial for obtaining reliable results:

• Genetic validation approaches:

  • Use siRNA-mediated knockdown of PLIN5 to confirm signal reduction

  • Compare signal between wild-type and PLIN5 knockout models

  • Overexpress PLIN5 and confirm corresponding signal increase

• Protein-level validation:

  • Perform Western blotting to confirm single band of expected molecular weight (51-55 kDa)

  • Compare antibody performance across multiple tissues with known PLIN5 expression patterns

  • Test antibody in various species if cross-reactivity is claimed

• Immunohistochemical validation:

  • Compare staining pattern with previously published results

  • Confirm expected subcellular localization (lipid droplet surface and mitochondrial association)

  • Verify tissue distribution (high in heart, oxidative skeletal muscle, brown adipose tissue)

• Control experiments:

  • Include primary antibody omission controls

  • Use pre-immune serum controls if available

  • Include isotype controls to assess non-specific binding

  • Use blocking peptide competition assays if available

• Cross-validation with other detection methods:

  • Compare FITC-conjugated antibody results with those obtained using unconjugated primary with secondary detection

  • Verify findings using antibodies targeting different epitopes of PLIN5

The antibody described in search result was validated in various tissues with known PLIN5 expression levels (heart, oxidative skeletal muscle, etc.) and showed the expected molecular weight and subcellular localization pattern.

How should PLIN5 localization patterns be interpreted in different experimental contexts?

Proper interpretation of PLIN5 staining patterns requires understanding its context-dependent localization:

• Normal physiological state:

  • In tissues with high fat oxidation capacity (heart, skeletal muscle): PLIN5 localizes to both lipid droplet surfaces and shows mitochondrial association

  • In lipid droplet-rich cells: PLIN5 predominantly surrounds lipid droplets

  • Expression levels highest in oxidative tissues (heart, oxidative skeletal muscle)

• During metabolic stress:

  • In MCDD (methionine-choline deficient diet) or high-fat diet conditions: PLIN5 expression increases in hepatic tissues

  • With hydrogen peroxide or LPS treatment: both mRNA and protein levels of PLIN5 increase significantly

  • With oleic acid treatment (inducing lipid droplet formation): PLIN5 strongly localizes to lipid droplet surfaces

• In pathological states:

  • NAFLD (non-alcoholic fatty liver disease): PLIN5 expression changes may serve as a survival strategy

  • Liver hepatocellular carcinoma (LIHC): PLIN5 has high expression, and low expression correlates with poor prognosis

  • During oxidative stress: PLIN5 upregulation via JNK-p38-ATF pathway occurs

• Colocalization interpretation:

  • PLIN5 and mitochondrial marker colocalization: indicates potential sites of lipid droplet-mitochondria interaction

  • PLIN5 and lipid droplet stain colocalization: confirms proper PLIN5 targeting to lipid droplets

  • Diffuse cytosolic PLIN5 staining: may indicate dysregulation or experimental artifacts

Research has shown that PLIN5's subcellular distribution is dynamic and can respond to metabolic changes, making it an informative marker for cellular lipid metabolism status .

How can researchers quantitatively analyze PLIN5 expression and localization data?

Quantitative analysis of PLIN5 expression and localization provides valuable insights into lipid metabolism regulation:

• Expression level quantification:

  • Western blot analysis with densitometry (expected molecular weight: 51-55 kDa)

  • qPCR for mRNA expression analysis

  • Flow cytometry using FITC-conjugated PLIN5 antibody for cell population analysis

• Localization pattern quantification:

  • Colocalization coefficient calculation between PLIN5 and organelle markers

  • Pearson's correlation coefficient for PLIN5 and mitochondrial markers

  • Manders' overlap coefficient for PLIN5 and lipid droplet markers

  • Distance analysis between PLIN5-positive structures and mitochondria

• Lipid droplet-associated analysis:

  • Quantify number and size of PLIN5-positive lipid droplets

  • Calculate percentage of lipid droplets with PLIN5 coating

  • Measure fluorescence intensity of PLIN5 on lipid droplet surfaces

  • Analyze correlation between PLIN5 intensity and lipid droplet size

• Software tools for analysis:

  • ImageJ/FIJI with colocalization plugins

  • CellProfiler for automated image analysis

  • Specialized confocal microscopy software packages

  • Custom analysis scripts for specific experimental needs

• Statistical approaches:

  • Compare PLIN5 expression/localization across different experimental conditions

  • Correlate PLIN5 metrics with functional outcomes (ROS levels, fatty acid oxidation rates)

  • Use appropriate statistical tests based on data distribution and experimental design

In research studies, PLIN5 expression changes have been quantified in response to various treatments (hydrogen peroxide, LPS, fatty acids) and correlated with functional outcomes like ROS levels and mitochondrial function .