PLIN3 Antibody

What is PLIN3 and what alternative nomenclature is used in the literature?

PLIN3 (Perilipin-3) is a 47 kDa intracellular transport protein belonging to the PAT (Perilipin/Adipophilin/TIP47) family of molecules. In scientific literature, you may encounter several alternative names for this protein:

• TIP47 (Tail Interacting Protein of 47 kDa)

• M6PRBP1 (Mannose 6-Phosphate Receptor Binding Protein 1)

• PP17 (Placental Protein 17)

• Cargo selection protein TIP47

The human PLIN3 protein consists of 434 amino acids and shares approximately 75% amino acid sequence identity with mouse and rat PLIN3 orthologs . When designing experiments or searching literature, consider using all these alternative names to ensure comprehensive coverage of relevant research.

What are the known structural features and isoforms of PLIN3?

PLIN3 contains a characteristic four-helix bundle domain that mediates interactions with cellular membranes. Key structural features include:

• Full-length form (isoform B): 434 amino acids with a calculated molecular weight of 47,075 Da

• Alternative isoform (isoform A): 251 amino acids with an alternate start site at position 184 of the full-length protein

• Post-translational modifications: The protein may undergo phosphorylation and acetylation

When performing Western blot analysis, PLIN3 typically appears as a band at approximately 47 kDa under reducing conditions . Understanding these structural characteristics is essential when selecting antibodies targeting specific epitopes or when interpreting experimental results showing multiple bands.

What are the primary cellular functions of PLIN3?

PLIN3 performs several critical cellular functions that make it an important target for research:

• Lipid droplet biology: Functions as a structural component required for the formation and maintenance of lipid storage droplets

• Intracellular trafficking: Essential for the transport of mannose 6-phosphate receptors (MPR) from endosomes to the trans-Golgi network

• Subcellular localization: Predominantly found in the cytoplasm, but also associates with:

  • Lipid droplet surfaces

  • Endosome membranes (peripheral membrane protein on the cytoplasmic side)

  • Lipid droplet envelopes and cores

  • Lipid "sails" structures

When designing experiments, consider these diverse functions and localizations to properly interpret PLIN3 detection patterns in different cellular compartments.

What criteria should guide my selection of a PLIN3 antibody for specific applications?

Selecting the appropriate PLIN3 antibody requires consideration of several factors:

For example, if studying specific isoforms, select an antibody targeting a region unique to that isoform. The search results show multiple antibodies targeting different regions, including AA 1-434 (full-length), AA 192-324 (internal region), AA 296-322 (C-terminal), and others .

How can I validate the specificity of my PLIN3 antibody?

Rigorous validation of antibody specificity is critical for reliable results:

• Positive control lysates: Test the antibody on cell lines known to express PLIN3, such as Daudi (human Burkitt's lymphoma), HeLa (human cervical epithelial carcinoma), and K562 (human chronic myelogenous leukemia) cell lines

• Western blot analysis: Confirm detection of a band at the expected molecular weight (~47 kDa)

• Knockout/knockdown controls: Compare staining between wild-type and PLIN3 knockout/knockdown samples

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate signal specificity

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm consistent detection patterns

For Western blot validation, recommended dilutions typically range from 1:2000 to 1:10000, though this varies by specific antibody .

What considerations are important regarding species cross-reactivity when using PLIN3 antibodies?

When working with different species, consider these cross-reactivity factors:

• Sequence homology: Human PLIN3 shares approximately 75% amino acid sequence identity with mouse and rat orthologs , which may affect antibody binding

• Epitope conservation: Verify that the epitope recognized by your antibody is conserved in your species of interest

• Validated species: Only use antibodies explicitly validated for your species of interest - search results show antibodies with validated reactivity to:

  • Human only

  • Human and mouse

  • Human, mouse, and rat

• Optimization: When using an antibody in a new species, perform careful titration and include appropriate controls

If working with non-mammalian models, additional validation steps are essential as cross-reactivity may be limited or unpredictable.

What are the optimal protocols for Western blot detection of PLIN3?

For successful Western blot detection of PLIN3, follow these methodological guidelines:

• Sample preparation:

  • Use RIPA or similar lysis buffer with protease inhibitors

  • Prepare samples under reducing conditions

  • Include phosphatase inhibitors if studying phosphorylated forms

• Gel selection and transfer:

  • 10-12% SDS-PAGE gels are appropriate for the 47 kDa protein

  • PVDF membranes show good results for PLIN3 detection

• Antibody dilutions:

  • Primary antibody: 1:2000-1:10000 dilution (optimize for each antibody)

  • Secondary antibody: HRP-conjugated anti-species IgG (typically 1:5000-1:10000)

• Detection:

  • PLIN3 appears as a specific band at approximately 47 kDa

  • Enhanced chemiluminescence (ECL) provides suitable detection sensitivity

• Controls:

  • Positive control: Lysates from Daudi, HeLa, or K562 cell lines

  • Loading control: Anti-GAPDH or anti-β-actin

As demonstrated in the R&D Systems data, using 0.25 μg/mL of Mouse Anti-Human Perilipin-3 Monoclonal Antibody followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody allows clear detection of PLIN3 in multiple cell lines .

How should I optimize immunohistochemistry and immunofluorescence experiments with PLIN3 antibodies?

For successful IHC and IF applications with PLIN3 antibodies:

• Fixation:

  • For frozen sections: 4% paraformaldehyde is typically effective

  • For paraffin sections: Standard formalin fixation with appropriate antigen retrieval

• Blocking and antibody dilutions:

  • IHC: Start with 1:20-1:200 dilution

  • IF: Start with 1:50-1:200 dilution

  • ICC: 1:100-1:500 dilution

• Antigen retrieval (for paraffin sections):

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Heat-induced epitope retrieval (HIER) methods typically work well

• Detection systems:

  • IHC: HRP/DAB or AP/Red detection systems

  • IF: Secondary antibodies conjugated to fluorophores appropriate for your microscopy setup

• Controls and counterstains:

  • Positive control: Tissues known to express PLIN3 (particularly lipid-rich tissues)

  • Counterstain: DAPI for nuclear visualization in IF

For mouse tissues, antibodies like GP37 (guinea pig polyclonal) have been successfully used at 1:100-1:200 dilution for frozen section IHC .

What technical considerations are important when detecting PLIN3 in different cellular compartments?

PLIN3 localizes to multiple cellular compartments, requiring careful experimental design:

• Lipid droplet localization:

  • Consider co-staining with lipid dyes (BODIPY, Nile Red) or other lipid droplet markers

  • Fasting or oleic acid loading may increase lipid droplet formation and enhance PLIN3 detection

  • Fixation must preserve lipid droplet integrity

• Endosomal localization:

  • Co-staining with endosomal markers (EEA1, Rab5, Rab7)

  • May require permeabilization optimization to maintain membrane structures

• Cytoplasmic vs. membrane association:

  • Differential extraction protocols can separate cytosolic and membrane-bound fractions

  • Membrane association occurs on the cytoplasmic side of endosomes

• Confocal microscopy:

  • Essential for accurate subcellular localization studies

  • Z-stack imaging to confirm co-localization with organelle markers

PLIN3 has been detected in lipid droplet envelopes, cores, and in structures called "lipid sails" , requiring careful microscopy techniques to distinguish these patterns.

How can I investigate PLIN3's role in lipid droplet dynamics?

For advanced studies on PLIN3's function in lipid metabolism:

• Lipid loading experiments:

  • Treat cells with oleic acid or other fatty acids to induce lipid droplet formation

  • Monitor PLIN3 recruitment to nascent lipid droplets using time-lapse microscopy

  • Compare recruitment kinetics with other PAT family proteins

• PLIN3 manipulation approaches:

  • siRNA/shRNA knockdown to assess necessity for lipid droplet formation

  • CRISPR/Cas9 knockout for complete elimination

  • Overexpression of wild-type or mutant forms to identify functional domains

• Co-immunoprecipitation studies:

  • Identify PLIN3 interaction partners during lipid droplet biogenesis

  • Compare interactome changes under different metabolic conditions

• Quantitative analysis methods:

  • Measure lipid droplet size, number, and PLIN3 coating using automated image analysis

  • Correlate PLIN3 levels with cellular lipid content measured biochemically

Given PLIN3's role as a structural component required for lipid droplet formation and maintenance , these approaches can elucidate its mechanistic contributions to lipid homeostasis.

What approaches can reveal PLIN3's functions in endosomal trafficking?

To investigate PLIN3's role in endosome-to-Golgi transport:

• Trafficking assays:

  • Monitor mannose 6-phosphate receptor trafficking using fluorescently tagged constructs

  • Pulse-chase experiments with endocytosed cargo

  • Live-cell imaging of PLIN3 and cargo movement

• Functional domain mapping:

  • Create deletion or point mutation constructs to identify regions essential for trafficking

  • Focus on the four-helix bundle thought to interact with membranes

• Interaction studies:

  • Investigate binding to RAB proteins that regulate vesicular transport

  • Examine interactions with membrane lipids and curvature-sensing domains

• Subcellular fractionation:

  • Isolate endosomal compartments to quantify PLIN3 association under different conditions

  • Compare wild-type versus trafficking-defective mutants

PLIN3 is required for the transport of mannose 6-phosphate receptors (MPR) from endosomes to the trans-Golgi network , making these approaches valuable for understanding its role in membrane trafficking pathways.

What are common troubleshooting strategies for PLIN3 antibody experiments?

When encountering issues with PLIN3 detection:

For Western blots specifically, PVDF membranes probed with 0.25 μg/mL of PLIN3 antibody followed by HRP-conjugated secondary antibody have shown consistent results with Daudi, HeLa, and K562 cell lines .

How should I interpret different expression patterns of PLIN3 across tissues and cell types?

When analyzing PLIN3 expression patterns:

• Tissue-specific expression:

  • PLIN3 expression varies across tissues based on metabolic activity and lipid storage requirements

  • Consider tissue-specific co-expression with other PAT family members

• Cell type heterogeneity:

  • Within tissues, different cell types may express PLIN3 at varying levels

  • Correlate expression with cellular lipid content or endocytic activity

• Physiological state influences:

  • Feeding/fasting status affects lipid droplet formation and PLIN3 recruitment

  • Developmental stages may show different expression patterns

• Standardized quantification:

  • Normalize expression to appropriate housekeeping genes

  • When comparing across tissues, consider using multiple normalization methods

• Validation across techniques:

  • Confirm expression patterns using complementary methods (WB, qPCR, IHC)

  • Single-cell approaches may reveal heterogeneity masked in bulk analysis

PLIN3 is particularly notable in milk fat globule membranes of human and bovine origin , highlighting its tissue-specific roles in specialized lipid structures.

What considerations are important when studying PLIN3 in disease models or patient samples?

For disease-focused PLIN3 research:

• Metabolic disorders:

  • Changes in PLIN3 expression may correlate with altered lipid metabolism

  • Compare expression across disease stages and treatment responses

• Cancer research:

  • PLIN3 has been detected in various cancer cell lines including Daudi, HeLa, and K562

  • Correlate expression with cancer progression or lipid metabolism alterations

• Technical considerations for patient samples:

  • Standardize collection, fixation, and processing protocols

  • Include appropriate age/sex-matched controls

  • Consider influences of medications or treatments on lipid metabolism

• Integration with other markers:

  • Analyze PLIN3 in context with other lipid metabolism markers

  • Consider co-localization with disease-specific markers

• Reproducibility and validation:

  • Use multiple antibodies targeting different epitopes

  • Validate key findings with functional studies when possible

When working with clinical samples, carefully optimize antibody dilutions based on the recommended ranges (IHC: 1:20-1:200, IF: 1:50-1:200) and include proper controls.

How can I quantitatively analyze PLIN3 distribution and association with cellular structures?

For quantitative analysis of PLIN3 localization:

• Image analysis approaches:

  • Measure co-localization coefficients with organelle markers

  • Quantify surface area or intensity of PLIN3-positive structures

  • Track dynamic changes using time-lapse imaging

• Biochemical fractionation:

  • Separate subcellular fractions (cytosol, lipid droplets, membranes)

  • Quantify PLIN3 distribution by Western blot

  • Assess changes in distribution following experimental manipulations

• Advanced microscopy techniques:

  • Super-resolution microscopy for detailed localization

  • FRET analysis for protein-protein interactions

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility studies

• Analysis software:

  • ImageJ with appropriate plugins for co-localization analysis

  • CellProfiler for automated high-throughput quantification

  • Custom analysis pipelines for specific structures

PLIN3 association with lipid droplets, endosome membranes, and other structures requires careful quantitative approaches to distinguish between these compartments and measure dynamic changes.