PLIN4 Antibody

What is PLIN4 and what applications can PLIN4 antibodies be used for?

PLIN4 (Perilipin 4) is a member of the perilipin family, a group of proteins that coat lipid droplets in adipocytes, the fatty tissue cells responsible for storing fat. These proteins are crucial regulators of lipid storage, and PLIN4 expression is typically elevated in obese animals and humans . PLIN4 is a lipid droplet protein (LDP) found predominantly in white adipose tissue, with lower expression in heart, skeletal muscle, and brown adipose tissue .

PLIN4 antibodies can be used for multiple research applications, including:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Enzyme-Linked Immunosorbent Assay (ELISA)

Most commercially available PLIN4 antibodies show reactivity with human samples, and some have cited reactivity with mouse and goat samples as well .

What are the recommended dilution ranges for PLIN4 antibodies in different applications?

Proper antibody dilution is critical for obtaining specific signals while minimizing background. Based on manufacturer recommendations, the following dilution ranges should be used for PLIN4 antibodies:

It is essential to note that optimal dilutions are application and sample-dependent. Researchers should perform a titration experiment in each testing system to determine the optimal antibody concentration for their specific application . For immunofluorescence studies, researchers have successfully used PLIN4 antibodies at 1:100 dilution for human skeletal muscle sections .

What is the molecular weight of PLIN4 and how does this affect antibody selection?

This variation in observed molecular weight should be considered when selecting antibodies and interpreting western blot results. Researchers should verify the specificity of their antibody by confirming the observed molecular weight matches the expected range for PLIN4.

What controls should be used when working with PLIN4 antibodies?

For PLIN4 antibody applications, researchers should include the following controls:

• Positive tissue/cell controls: PC-3 cells and Jurkat cells have been validated as positive controls for western blot applications, while human pancreas tissue has been verified for immunohistochemistry .

• Fractionation controls: When analyzing subcellular localization, separate the sample into cytosolic, membrane, nuclear, and cytoskeleton fractions. Silver staining of SDS-PAGE gels can be used to confirm equal protein loading across fractions .

• Marker proteins: For co-localization studies, include markers for subcellular compartments. For example, dystrophin can be used as a plasma membrane marker when studying PLIN4 localization near the sarcolemma .

How is PLIN4 subcellularly localized in skeletal muscle, and what are the optimal methods for detection?

PLIN4 demonstrates a unique subcellular localization pattern in skeletal muscle that differs from other perilipin family members. Several lines of evidence from microscopy and fractionation studies indicate that PLIN4 is primarily associated with the sarcolemma (plasma membrane) or found in the subsarcolemmal (SS) region .

For optimal detection of PLIN4's subcellular localization:

• Immunofluorescence microscopy approach:

  • Prepare 4-μm sections using a rotary microtome

  • Mount on Superfrost Plus glass slides and incubate overnight at 37°C

  • Deparaffinize in xylene (2 × 5 min)

  • Rehydrate stepwise in 100%, 96%, 70%, and 50% ethanol (5 min each)

  • Perform heat-induced antigen retrieval with 1 mmol/L EDTA (pH 8) at 95°C for 15 min

  • Block with 0.01 mol/L PBS containing 0.05% Triton X-100, 1% BSA, and 3% newborn calf serum

  • Incubate with primary antibodies overnight at 4°C (PLIN4 1:100 and dystrophin 1:100 as a plasma membrane marker)

  • Incubate with fluorochrome-conjugated secondary antibodies (1:400)

• Western blotting with subcellular fractionation:

  • Homogenize and fractionate muscle samples (approximately 50 mg)

  • Separate into cytosolic, membrane, nuclear, and cytoskeleton fractions

  • Perform SDS-PAGE (7.5%) followed by transfer to PVDF membrane

  • Block overnight at 4°C in TBS-T containing 3% BSA and 0.02% sodium azide

  • Incubate with anti-PLIN4 antibody overnight at 4°C

  • Visualize with enhanced chemiluminescence after HRP-conjugated secondary antibody incubation

Using these approaches, researchers can confirm that PLIN4 is located at or close to the sarcolemma, in contrast to PLIN5 which shows a uniform distribution throughout the muscle fiber .

How does PLIN4 expression correlate with lipid droplet populations in skeletal muscle, and what are the implications for metabolism research?

Research has revealed significant correlations between PLIN4 expression and specific lipid droplet (LD) populations in skeletal muscle. Notably, there is a strong and significant correlation between changes in PLIN4 mRNA expression and changes in subsarcolemmal (SS) lipid droplet area (r = 0.58, P = 0.01) following long-term physical activity . In contrast, no significant correlation was observed between PLIN4 mRNA and intramyofibrillar (IMF) lipid droplets (r = 0.15, P = 0.53) .

Considering absolute values, researchers found a trend toward positive correlation between PLIN4 mRNA and the area of SS LDs both before and after 12 weeks of training (r = 0.44, P = 0.07, and r = 0.41, P = 0.09, respectively) . Additionally, PLIN4 expression positively correlated with IMF LD area before training (r = 0.47, P < 0.05) but showed only a trend after the training period (r = 0.40, P = 0.09) .

For metabolism researchers, these correlations suggest that:

• PLIN4 is preferentially associated with SS LDs rather than IMF LDs

• SS LDs appear more metabolically responsive to physical activity than IMF LDs

• PLIN4 reduction following long-term training is specifically linked to decreases in SS LD size

These findings highlight the importance of analyzing distinct LD populations separately when studying the effects of interventions on skeletal muscle lipid metabolism .

What are the technical challenges in PLIN4 antibody-based research, and how can they be addressed?

Several technical challenges arise when working with PLIN4 antibodies for research applications:

• Multiple protein bands: PLIN4 may appear as multiple bands in western blots, typically around 100-120 kDa . To address this:

  • Include positive control samples (PC-3 cells, Jurkat cells)

  • Use subcellular fractionation to confirm band specificity in membrane/organelle fractions

  • Verify bands using multiple antibodies against different epitopes of PLIN4

• Antigen retrieval optimization: For immunohistochemistry, proper antigen retrieval is critical. Recommended approaches include:

  • Primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • For immunofluorescence on muscle sections: 1 mmol/L EDTA (pH 8) at 95°C for 15 min

• Antibody validation across species: While PLIN4 antibodies have been tested in human samples, cited reactivity includes mouse and goat samples . When working with non-human samples:

  • Verify antibody specificity with appropriate positive and negative controls

  • Consider antibodies raised against conserved epitopes

  • The PLIN4 antibody with catalog number ABIN2613464 shows cross-reactivity with cow, dog, mouse, pig, and rat

• Storage and stability considerations: To maintain antibody performance:

  • Store at -20°C

  • Antibodies are typically stable for one year after shipment

  • Aliquoting may not be necessary for -20°C storage

  • Some preparations contain 0.1% BSA in smaller (20μl) sizes

How does PLIN4 expression change in response to exercise, and what methodologies can detect these changes?

Long-term physical activity has been shown to reduce PLIN4 expression in skeletal muscle . This reduction correlates with decreased lipid droplet size in the subsarcolemmal region, linking PLIN4 reduction to changes in lipid storage patterns following exercise .

To effectively study exercise-induced changes in PLIN4 expression, researchers can employ several methodological approaches:

• mRNA expression analysis:

  • Extract RNA from muscle biopsies before and after exercise intervention

  • Perform RT-qPCR to quantify changes in PLIN4 mRNA expression

  • Compare these changes with other PLIN family members (PLIN2, PLIN3, PLIN5)

  • Correlate expression changes with physiological parameters (e.g., VO2max, insulin sensitivity)

• Protein quantification:

  • Perform western blotting on whole muscle lysates or fractionated samples

  • Use the recommended antibody dilutions (1:500-1:2000)

  • Quantify band intensity using appropriate software

  • Normalize to loading controls or reference proteins

• Microscopic analysis of lipid droplets and PLIN4 co-localization:

  • Perform immunofluorescence co-staining of PLIN4 with lipid droplet markers

  • Use dystrophin as a plasma membrane marker

  • Quantify changes in co-localization patterns following exercise

  • Separately analyze SS and IMF regions to detect region-specific changes

These methodologies allow researchers to comprehensively assess how exercise affects PLIN4 expression and its relationship with lipid storage dynamics in skeletal muscle.