Perlwapin Antibody

What are Perilipin proteins and why are they important research targets?

Perilipin proteins are a family of lipid droplet-associated proteins that play crucial roles in lipid metabolism. Perilipin (also known as lipid droplet-associated protein) localizes on the surface of intracellular lipid droplets within adipocytes, where it serves as a protective barrier for stored lipids until they are mobilized by hormone-sensitive lipase (HSL) . This localization allows Perilipin to regulate adipocyte lipid metabolism, making it an essential protein for maintaining energy homeostasis. Dysregulation of Perilipin has been associated with obesity, where elevated levels can lead to increased fat storage . The critical role of Perilipins in lipid metabolism regulation makes them important targets for research related to metabolic disorders, obesity, and energy homeostasis.

What are the different types of Perilipin proteins that can be detected with antibodies?

There are several types of Perilipin proteins that researchers study, with Perilipin-1 and Perilipin-5 being among the most commonly investigated:

• Perilipin-1: Encoded by the PLIN1 gene in humans, this protein has an amino acid length of 522 and an expected mass of 56 kDa. It may also be known as Peri, Perilipin A, Plin, FPLD4, and lipid droplet-associated protein . Perilipin-1 is primarily expressed in adipocytes.

• Perilipin-5: Also known as LSDP5 or OXPAT, this lipid droplet coat protein promotes the association of lipid droplets with mitochondria. It is mainly present in tissues with high fat-oxidative capacity, such as heart, skeletal muscles, and brown adipose tissue . Its calculated molecular weight is approximately 51 kDa, though the observed molecular weight in experiments is typically 51-55 kDa .

What tissue samples are suitable for Perilipin antibody applications?

Based on validated research applications, Perilipin antibodies have shown reactivity with various tissue samples:

For Perilipin-1 antibodies:

• Human adipose tissue

• Mouse adipose tissue

• Rat adipose tissue

• Various cell lines including MCF-7 cells

For Perilipin-5 antibodies:

• Mouse liver tissue

• Pig heart tissue

• Mouse heart tissue

• Rat heart tissue

• Human liver tissue

• Mouse brown adipose tissue

• Human heart tissue

This tissue distribution reflects the biological role of different Perilipin family members, with Perilipin-1 predominantly expressed in adipose tissue and Perilipin-5 in tissues with high oxidative capacity.

What are the recommended applications and dilutions for Perilipin antibodies?

Perilipin antibodies can be used in multiple experimental applications. Based on validated data, here are the recommended applications and dilutions:

For Perilipin-5 antibody (26951-1-AP):

Note: Optimal dilutions may be sample-dependent and should be determined for each specific experimental setup .

For Perilipin Antibody (G-2):
This antibody can be used for western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry with paraffin-embedded sections (IHCP), and enzyme-linked immunosorbent assay (ELISA) .

How can I optimize antigen retrieval for Perilipin immunohistochemistry?

For optimal results in immunohistochemistry applications with Perilipin antibodies, the following antigen retrieval methods are recommended:

• Primary suggestion: Antigen retrieval with TE buffer at pH 9.0

• Alternative method: Antigen retrieval with citrate buffer at pH 6.0

The choice between these methods may depend on the specific tissue being analyzed and should be optimized for each experimental context. Proper antigen retrieval is crucial for exposing epitopes that may be masked during fixation processes, particularly in formalin-fixed, paraffin-embedded tissues.

What conjugates are available for Perilipin antibodies and how should they be selected?

Perilipin antibodies are available in various conjugated forms to accommodate different experimental needs:

Available conjugates include:

• Unconjugated (native antibody)

• Horseradish peroxidase (HRP)

• Fluorescent tags (FITC, PE)

• Alexa Fluor® conjugates

• Agarose conjugates (for immunoprecipitation)

Selection criteria should include:

• The detection method of your experiment (chromogenic vs. fluorescent)

• The sensitivity required

• The availability of appropriate imaging equipment

• Potential for multiplexing with other antibodies

• Background concerns in the specific tissue/application

How can antibody sensitivity and specificity for Perilipin detection be enhanced?

Enhancing antibody sensitivity and specificity is crucial for obtaining reliable results, especially when working with complex tissue samples. A novel approach involving peptide cross-linking with cupric ions at high pH has been developed and may improve some antibody performances:

This technique has been shown to improve the affinity and lower cross-reactivity with non-specific bands for approximately 20% of antibodies tested (5 out of 25) . The improvement is mediated by copper ions, which form stable coordination complexes with the amide of peptide bonds and various amino acid side-chain residues.

Potential mechanisms for this enhancement include:

• Copper-peptide interactions that may improve reactivity and specificity

• Coordination and blocking of 'non-specific' sites

• Improved epitope binding

What role does Perilipin phosphorylation play in functional studies, and how can phospho-specific antibodies be utilized?

Perilipin undergoes important post-translational modifications, particularly phosphorylation, which enhances its sensitivity to hormone-sensitive lipase (HSL), playing a pivotal role in the lipolytic process . For researchers studying the functional aspects of Perilipin:

• Phosphorylation significance: When Perilipin is phosphorylated (typically by protein kinase A during lipolytic stimulation), it changes conformation allowing lipases to access the stored triglycerides, thus regulating the rate of lipolysis.

• Phospho-specific antibodies: These specialized antibodies recognize only the phosphorylated form of Perilipin and can be valuable tools for:

  • Tracking the activation state of Perilipin in response to hormonal stimulation

  • Comparing phosphorylation levels between different experimental conditions

  • Evaluating the effects of drugs or genetic manipulations on Perilipin activity

• Experimental approaches: When using phospho-specific antibodies, researchers should consider:

  • Including appropriate controls (phosphatase-treated samples)

  • Timing sample collection to capture the relevant phosphorylation events

  • Using complementary methods (such as Phos-tag gels) to validate phosphorylation status

What are the key considerations for validating Perilipin antibodies in knockout/knockdown models?

Antibody validation using genetic models is essential for confirming specificity. Based on the search results, Perilipin-5 antibodies have been validated in knockout/knockdown models in at least two published studies . When performing such validations:

• Experimental design considerations:

  • Use appropriate genetic controls (complete knockout, conditional knockout, or siRNA/shRNA knockdown)

  • Include wild-type samples processed in parallel

  • Consider tissue-specific expression patterns when selecting validation tissues

  • Examine multiple dilutions of the antibody to assess sensitivity and specificity

• Validation criteria:

  • Complete absence or significant reduction of the target band/signal in knockout/knockdown samples

  • Consistent molecular weight detection (e.g., 51-55 kDa for Perilipin-5)

  • Comparable results across multiple applications (WB, IHC, IF) if the antibody is intended for multiple uses

  • Secondary validation using an alternative antibody targeting a different epitope

• Troubleshooting inconsistencies:

  • If signals persist in knockout models, investigate potential cross-reactivity with other Perilipin family members

  • Consider compensation mechanisms that might upregulate related proteins

  • Evaluate whether the knockout is complete or if truncated proteins are still expressed

What are the optimal storage conditions for Perilipin antibodies to maintain reactivity?

Proper storage of antibodies is crucial for maintaining their reactivity and specificity over time. Based on the manufacturer's recommendations for Perilipin antibodies:

• Storage temperature: Store at -20°C for long-term stability

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 provides optimal stability

• Aliquoting requirements: For smaller sizes (e.g., 20μl), aliquoting may be unnecessary for -20°C storage

• Stability period: When properly stored, antibodies are typically stable for one year after shipment

Additional storage recommendations to maintain antibody performance include avoiding repeated freeze-thaw cycles, storing in small aliquots if frequent use is anticipated, and keeping antibodies away from light if they are conjugated to fluorophores.

How can cross-reactivity issues between different Perilipin family members be addressed?

Cross-reactivity between Perilipin family members can complicate experimental interpretation due to their structural similarities. Researchers should consider the following strategies:

• Epitope selection: Choose antibodies raised against unique regions of the target Perilipin protein rather than conserved domains

• Validation in multiple systems:

  • Test antibodies in tissues with differential expression of Perilipin family members

  • Validate using overexpression systems with individual Perilipin proteins

  • Confirm specificity using knockout/knockdown models for the target Perilipin

• Experimental controls:

  • Include recombinant protein standards for precise molecular weight comparisons

  • Use tissues known to express specific Perilipin family members preferentially (e.g., adipose tissue for Perilipin-1, heart tissue for Perilipin-5)

• Analytical approaches:

  • Perform pre-absorption tests with specific peptides to confirm epitope specificity

  • Use competing peptides to evaluate binding specificity

  • Consider using multiple antibodies targeting different epitopes of the same protein

What are the common pitfalls in quantitative analysis of Perilipin expression using antibody-based methods?

Quantitative analysis of Perilipin expression can be challenging due to several factors that researchers should be aware of:

• Technical considerations for Western blotting:

  • Loading control selection: Traditional housekeeping proteins may not be appropriate for all tissues, especially when comparing tissues with vastly different lipid content

  • Linear range determination: Establish the linear range of detection for both the Perilipin antibody and loading control antibodies

  • Signal saturation: Avoid overexposure which can lead to inaccurate quantification

• Immunohistochemistry/Immunofluorescence challenges:

  • Lipid droplet size variation: Perilipin coats lipid droplets of varying sizes, potentially affecting signal intensity independent of expression level

  • Section thickness consistency: Maintain uniform section thickness for accurate comparisons

  • Autofluorescence: Lipid-rich tissues often exhibit significant autofluorescence that must be accounted for

• Biological variables affecting interpretation:

  • Nutritional status: Fasting/feeding can dramatically alter Perilipin localization and expression

  • Tissue heterogeneity: Different cell populations within the same tissue may have varying Perilipin expression levels

  • Pathological conditions: Disease states can alter Perilipin expression and localization independent of experimental variables

• Data normalization approaches:

  • For Western blots: Normalize to total protein (using stain-free technology or total protein stains) rather than single loading control proteins

  • For microscopy: Consider normalizing to tissue area, cell number, or lipid droplet content depending on the research question

How can Perilipin antibodies be effectively used in studies of adipocyte differentiation?

Perilipin antibodies are valuable tools for monitoring adipocyte differentiation as Perilipin expression increases dramatically during this process:

• Temporal expression analysis:

  • Use Perilipin-1 antibodies to track the progression of preadipocyte to mature adipocyte conversion

  • Combine with other differentiation markers (such as PPARγ or C/EBPα) for comprehensive analysis

  • Monitor both protein levels (by Western blot) and subcellular localization (by immunofluorescence)

• Experimental approaches:

  • Time-course studies: Collect samples at defined intervals during differentiation to create expression profiles

  • Perturbation analysis: Evaluate how genetic or pharmacological interventions affect Perilipin expression during differentiation

  • Co-localization studies: Combine Perilipin staining with lipid dyes (e.g., BODIPY, Oil Red O) to correlate protein expression with lipid accumulation

• Quantitative assessment methods:

  • Measure percentage of Perilipin-positive cells as an index of differentiation efficiency

  • Quantify Perilipin signal intensity per cell as a measure of differentiation degree

  • Analyze lipid droplet size and number in relation to Perilipin coating as indicators of adipocyte maturity

What considerations are important when using Perilipin antibodies in studies of lipid metabolism disorders?

When investigating lipid metabolism disorders such as obesity, fatty liver disease, or lipodystrophy, several factors should be considered when using Perilipin antibodies:

• Isoform-specific expression changes:

  • Different Perilipin family members may be differentially affected in various disorders

  • Perilipin-1 is predominantly expressed in adipose tissue and may be altered in obesity

  • Perilipin-5 is expressed in tissues with high oxidative capacity (heart, skeletal muscle, liver) and may be relevant in non-alcoholic fatty liver disease or cardiac lipotoxicity

• Tissue-specific considerations:

  • For liver studies: Normal hepatocytes have minimal Perilipin expression, but this increases with steatosis

  • For skeletal muscle: Distribution may be fiber-type dependent

  • For adipose tissue: Different adipose depots (subcutaneous vs. visceral) may show different expression patterns

• Methodological approaches:

  • Consider dual staining with other markers of metabolic dysfunction

  • Combine protein expression analysis with functional lipid metabolism assays

  • Evaluate phosphorylation status as an indicator of functional activity in disease states

How can multi-parametric analysis be conducted using Perilipin antibodies in combination with other markers?

Multi-parametric analysis combining Perilipin antibodies with other markers can provide comprehensive insights into lipid metabolism and related processes:

• Immunofluorescence multiplexing strategies:

  • Combine Perilipin antibodies with antibodies against other proteins involved in lipid metabolism (e.g., ATGL, HSL, CGI-58)

  • Use differently conjugated secondary antibodies or directly conjugated primary antibodies to avoid spectral overlap

  • Include cellular compartment markers (mitochondria, ER) to study organelle interactions with lipid droplets

• Flow cytometry applications:

  • Perilipin antibodies conjugated to fluorophores can be used in flow cytometry for quantitative analysis of heterogeneous cell populations

  • Combined with other markers, this approach allows identification and characterization of specific cell subpopulations based on their lipid storage properties

• Advanced imaging techniques:

  • Super-resolution microscopy can reveal detailed spatial relationships between Perilipin and other proteins at the lipid droplet surface

  • Live-cell imaging using fluorescently tagged Perilipin antibody fragments can monitor dynamic processes

  • Correlative light and electron microscopy can connect Perilipin localization with ultrastructural features