Patatin-07 Antibody

What is PNPLA7 and why are specific antibodies needed for its detection?

PNPLA7, also known as neuropathy target esterase-related esterase (NRE), is a patatin-like phospholipase that functions as a lysophospholipase with preference for lysophosphatidylcholine (LPC). This protein plays significant roles in:

• Metabolic regulation (responds to fasting/feeding cycles in skeletal muscle)

• Macrophage polarization and inflammatory responses

• Lipid metabolism, particularly lysophospholipid degradation

Specific antibodies are essential for PNPLA7 detection because:

• PNPLA7 has multiple isoforms and potential post-translational modifications

• It shares sequence homology with other PNPLA family members, risking cross-reactivity

• Its expression is tissue-specific and regulated by metabolic conditions

• Standard detection requires antibodies validated for specific applications (WB, IHC, IF)

How can I validate the specificity of a PNPLA7 antibody for my research?

Multiple validation approaches should be employed to ensure antibody specificity:

Antibody Neutralization Method:

• Incubate primary antibody with excess PNPLA7 peptide antigen (use PrEST Antigen if available)

• Prepare a mixture containing antibody (0.4 μg/mL), PNPLA7 PrEST antigen (0.8 μg/mL), and buffer with 1M urea

• Incubate for 3.5 hours at 22°C with constant shaking (300 rpm)

• Dilute with primary antibody buffer to working concentration

• Compare immunoblots using neutralized versus non-neutralized antibody

Gene Silencing Validation:

• Treat cells with siRNA against PNPLA7 or scrambled control

• Collect samples 72h post-transfection

• Perform Western blot to confirm reduced signal intensity at expected molecular weight (~150 kDa)

• Calculate and compare band densitometry between siPNPLA7 and siSCR samples

In silico Analysis:

• Use BLAST to check antibody epitope sequence for alignment with other proteins

• Common PNPLA7 antibody epitope sequence: CEVGYQHGRTVFDIWGRSGVLEKMLRDQQGPSKKPASAVLTCPNASFTDLAEIVSRIEPAKPAMVDDESDYQTEYEEELLDVPRDAYADFQSTSAQQGSDLEDESSLRHRHPSLAFPKLSE

Which PNPLA7 immunoreactive bands are specifically associated with the target protein?

When using PNPLA7 antibodies like HPA009130, researchers should be aware of multiple immunoreactive bands:

Important Note: The ~150 kDa band is the most reliable indicator of PNPLA7 expression in human skeletal muscle tissue and cultured myotubes. The ~225 kDa band, while specifically binding the PNPLA7 antibody, is not affected by PNPLA7 gene silencing, suggesting it may represent a protein complex with different turnover kinetics or cross-reactivity .

What is the optimal Western blot protocol for detecting PNPLA7 in tissue samples?

Based on published methodologies, the following optimized protocol yields consistent PNPLA7 detection:

Sample Preparation:

• Homogenize tissue in Laemmli buffer

• For muscle tissue, use protocols established for human semitendinosus muscle

Electrophoresis and Transfer:

• Standard SDS-PAGE (note: potential dimers dissociate in SDS-containing media)

• Transfer to PVDF/nitrocellulose membrane

• Stain with Ponceau S to evaluate sample loading and transfer

Blocking and Antibody Incubation:

• Block membrane with 7.5% (w/v) dry milk in TBST (20 mM Tris, 150 mM NaCl, 0.02% (v/v) Tween-20, pH=7.5) with addition of 0.8% BSA for 1-2 hours at room temperature

• Wash three times with TBST

• Incubate with primary antibody (recommended dilution 1:500) in primary antibody buffer (20 mM Tris, 150 mM NaCl, 0.1% (w/v) BSA, 0.1% (w/v) sodium azide, pH=7.5) overnight at 4°C

• Wash with TBST three times for 10 minutes

• Incubate with HRP-conjugated secondary antibody with 5% (w/v) dry milk in TBST for one hour at room temperature

• Wash in TBST three times for 10 minutes

• Incubate with ECL reagent for one minute and visualize

Controls to Include:

• Actin as loading control

• Neutralized antibody control

• Positive control (human skeletal muscle or cells with confirmed PNPLA7 expression)

How can I resolve contradictory results between different PNPLA7 antibodies?

Discrepancies between antibodies targeting the same protein are common in research. For PNPLA7 specifically:

Systematic Troubleshooting Approach:

• Compare epitope sequences:

  • Request epitope information from manufacturers

  • Different epitopes may detect different isoforms or post-translationally modified versions

  • Check for epitope masking in your experimental conditions

• Perform cross-validation experiments:

  • Test all antibodies on the same positive and negative control samples

  • Include antibody neutralization controls for each antibody

  • Perform gene silencing to verify specificity

• Evaluate antibody characteristics:

  • Compare polyclonal vs. monoclonal antibodies (polyclonals may detect multiple epitopes)

  • Check species reactivity (human vs. mouse PNPLA7 show differences)

  • Consider antibody format (full IgG vs. Fab fragments)

• Structural considerations:

  • PNPLA7 may undergo conformational changes affecting epitope accessibility

  • Consider induced-fit binding (S1/S2) vs. pre-existing conformations (S3)

  • Some antibodies may recognize only specific structural states

Recommendation: When publishing contradictory results, document all validation steps performed and provide clear rationale for antibody selection based on the specific research question .

What methods can I use to study PNPLA7's function in macrophage polarization?

PNPLA7 plays a significant role in macrophage polarization, particularly in suppressing pro-inflammatory M1 responses. Based on recent research, the following methodologies are recommended:

Expression Analysis During Polarization:

• Treat RAW264.7 macrophages or BMDMs with LPS (100 ng/mL) to induce M1 polarization

• Collect samples at multiple timepoints (0h, 4h, 8h, 24h)

• Measure PNPLA7 mRNA expression by RT-qPCR

• Quantify protein levels by Western blot using validated antibodies

Genetic Manipulation Approaches:

• Overexpression:

  • Generate stable cell lines expressing PNPLA7-GFP vs. GFP controls

  • Confirm expression by immunoblotting with anti-GFP antibody

  • Challenge with LPS and measure inflammatory markers

• Knockdown:

  • Use siRNA or shRNA targeting PNPLA7

  • Confirm knockdown efficiency by qPCR and Western blot

  • Challenge with LPS and measure inflammatory responses

Downstream Pathway Analysis:
Monitor key regulatory pathways affected by PNPLA7 manipulation:

• SIRT1 mRNA and protein levels

• NF-κB p65 acetylation status

• Phosphorylated p38 MAPK levels

• Pro-inflammatory gene expression (IL-1β, IL-6, iNOS, TNF-α)

Table: Effect of PNPLA7 Manipulation on Inflammatory Signaling

These methodologies provide a comprehensive approach to studying PNPLA7's immunomodulatory functions in macrophage polarization and inflammatory responses .

How should I design experiments to investigate PNPLA7 regulation by metabolic conditions?

PNPLA7 expression is influenced by nutritional status, particularly in metabolic tissues. The following experimental design considerations are crucial:

In Vitro Metabolic Models:

• Insulin and glucose regulation:

  • Culture myotubes in serum- and insulin-free DMEM with variable glucose concentrations (0.5 g/L, 1 g/L, 4.5 g/L)

  • Treat with insulin (0.1 μg/mL and/or 10 μg/mL) for 16h

  • Analyze PNPLA7 mRNA and protein expression

• cAMP pathway involvement:

  • Treat myotubes with dibutyryl-cAMP (200 μM) or forskolin (5 μM)

  • Include appropriate vehicle controls

  • Examine effects on PNPLA7 expression and activity

• Nutrient deprivation models:

  • Compare standard vs. low lipid content media

  • Monitor changes in PNPLA7 expression

  • Analyze downstream effects on lipid profiles (FFA, DAG, TAG)

Sample Analysis:

• Quantify PNPLA7 mRNA using RT-qPCR

• Measure protein levels by Western blot

• Perform lipidomic analyses to assess changes in lysophospholipid metabolism

• Consider subcellular fractionation to detect changes in PNPLA7 localization

Statistical Analysis:

• Present data as means ± SEM

• Use t-test for simple comparisons

• Apply two-way ANOVA with Tukey post hoc test for multiple variables

• Consider p ≤ 0.05 as statistically significant

What approaches can be used to study PNPLA7's enzymatic activity in different tissue samples?

Investigating PNPLA7's enzymatic function requires specialized approaches beyond simple expression analysis:

Enzymatic Activity Assays:

• Lysophospholipase activity:

  • Measure hydrolysis of lysophosphatidylcholine (LPC) to generate glycerophosphocholine and fatty acids

  • Monitor released fatty acids by colorimetric or HPLC methods

  • Compare activity with specific inhibitors to confirm specificity

• Substrate specificity determination:

  • Test activity against various substrates: LPC, phosphatidylcholine, triglycerides, monoacylglycerols

  • Confirm PNPLA7 preference for lysophospholipids over other lipid classes

Lipidomic Analysis for Functional Validation:

• Separate neutral and total phospholipid groups via thin-layer chromatography (TLC)

• Quantify individual lipid classes: TAG, FFA, DAG, phospholipids

• Determine fatty acid composition for each lipid class

• Compare lipid profiles between:

  • PNPLA7 wildtype vs. knockdown/knockout models

  • Different metabolic conditions (fed vs. fasted state)

  • Various tissues (skeletal muscle, adipose, macrophages)

Subcellular Localization:

• Perform subcellular fractionation to isolate mitochondria, cytosol, and other compartments

• Measure PNPLA7 distribution across fractions

• Correlate localization with site-specific activity

• Use immunofluorescence with validated antibodies to visualize distribution

Important observation: Recent studies indicate PNPLA7 may specifically degrade phosphatidylglycerol (PG) to generate lysobisphosphatidic acid (LBPA) in mitochondria, suggesting tissue-specific functions that should be considered in experimental design .

How can I produce and validate custom antibodies against PNPLA7?

For researchers requiring custom antibodies with specific properties, the following approaches have proven successful:

Antigen Selection Strategies:

• PLP domain expression:

  • Amplify PLP domain from cDNA

  • Insert into expression vector with C-terminal HIS tag

  • Express using cell-free systems (e.g., wheat germ)

  • Purify by nickel column chromatography

• Peptide synthesis approach:

  • Design multiple peptides from unique locations along PNPLA7

  • Maximize estimated immunogenicity

  • Minimize cross-reactivity with other PNPLA family members

  • Conjugate to KLH (keyhole limpet hemocyanin)

Immunization Protocol:

• Mix 100 μg of purified protein or peptide-KLH conjugate with Freund's complete adjuvant (50:50)

• Inject subcutaneously into mice/rabbits

• Administer booster intradermal injections (100 μg antigen in Freund's incomplete adjuvant) at 4 and 8 weeks

• Collect serum by cardiac puncture 2 weeks after final booster

Validation Requirements:

• Cross-reactivity testing against other PNPLA family members

• Comparison with commercial antibodies

• Testing across multiple applications (WB, IHC, IF)

• Confirmation using knockout/knockdown models

How do I design experiments to study structure-function relationships of PNPLA7 using antibodies?

Understanding PNPLA7's structural properties in relation to its function requires specialized approaches:

Epitope Mapping Strategies:

• Generate panel of antibodies targeting different domains

• Use truncated or mutated PNPLA7 constructs to identify binding regions

• Perform competitive binding assays to determine overlapping epitopes

• Consider X-ray crystallography for high-resolution analysis of antibody-antigen interactions

Structural Classification Approaches:
Based on binding surface changes upon antigen binding, PNPLA family antibodies can be classified into:

• S1: Creation of a pocket on binding surface

• S2: Removal of a pocket from binding surface

• S3: No apparent change in binding sites

This classification helps understand structural dynamics and can guide antibody selection for specific applications .

Conformational Analysis:

• Calculate RMSD differences between free and bound antibody forms

• Analyze variable vs. constant domain movements

• Examine heavy and light chain packing

• Monitor CDR loop conformational changes

Table: Structural Classification of Antibody-Antigen Interactions

Understanding these structural classifications can inform proper antibody selection for specific experimental applications related to PNPLA7 research .

What strategies can resolve issues with non-specific binding or inconsistent PNPLA7 antibody performance?

When encountering issues with PNPLA7 antibodies, systematic troubleshooting can identify and resolve problems:

Protocol Optimization:

• Blocking conditions:

  • Increase blocking agent concentration (7.5% dry milk)

  • Add BSA (0.8%) to reduce non-specific binding

  • Extend blocking time to 2 hours at room temperature

• Antibody dilution optimization:

  • Test serial dilutions (1:50 to 1:1000) to determine optimal concentration

  • Reduce primary antibody concentration if background is high

  • Consider overnight incubation at 4°C rather than shorter times at room temperature

• Washing stringency:

  • Increase number of washes (minimum 3× for 10 minutes each)

  • Adjust Tween-20 concentration in wash buffer (0.02% to 0.1%)

  • Consider alternative wash buffers for problematic samples

Sample-Related Considerations:

• Check sample integrity and protein degradation

• Evaluate potential post-translational modifications affecting epitope recognition

• Consider tissue-specific expression patterns and potential isoforms

• Be aware of potential dimers or complex formation affecting band patterns

Advanced Troubleshooting:

• Perform parallel testing with multiple antibodies targeting different epitopes

• Include controls for non-specific binding (secondary antibody only, isotype controls)

• Consider pre-absorption of antibody with related proteins to improve specificity

• For phosphorylated targets, include phosphatase treatments as controls

How can PNPLA7 antibodies be used to study connections between lipid metabolism and inflammation?

Recent research reveals PNPLA7's dual role in lipid metabolism and inflammatory responses, offering new research directions:

Integrated Experimental Approaches:

• Macrophage polarization studies:

  • Compare PNPLA7 expression in naïve, M1, and M2 macrophages

  • Monitor changes in lipid composition during polarization using lipidomics

  • Correlate PNPLA7 activity with inflammatory marker expression

• Metabolic challenge models:

  • Expose macrophages to lipid-rich environments

  • Examine PNPLA7's role in lipid handling and inflammatory responses

  • Test if PNPLA7 overexpression protects against lipid-induced inflammation

• Signaling pathway investigations:

  • Study how PNPLA7-mediated lipid metabolism affects SIRT1-NF-κB axis

  • Investigate connections between LPC metabolism and inflammatory signaling

  • Explore how fatty acid release by PNPLA7 impacts cellular functions

Potential Mechanisms:

• PNPLA7's lysoPC hydrolase activity may reduce pro-inflammatory lysophospholipids

• PNPLA7-generated fatty acids might serve as PPAR ligands to resolve inflammation

• PNPLA7 activity could alter membrane composition affecting signaling platform function

What are the latest methodological advances in studying PNPLA7 interactions with other proteins?

Understanding PNPLA7's protein-protein interactions requires specialized techniques:

Proximity-Based Approaches:

• BioID or TurboID proximity labeling to identify proteins in close proximity to PNPLA7

• FRET/BRET assays to study dynamic interactions in living cells

• Split-GFP complementation to visualize direct protein interactions

• Co-immunoprecipitation with PNPLA7 antibodies followed by mass spectrometry

Antibody-Based Interaction Mapping:

• Use epitope-specific antibodies to block potential interaction interfaces

• Develop antibodies against known or predicted interaction domains

• Apply antibody-based proximity ligation assays (PLA) to visualize interactions in situ

• Employ antibody competition assays to map binding sites

Graph Convolutional Model Applications:
Recent computational approaches can predict protein-protein interactions based on:

• Interface structure analysis

• Iterative mutation optimization strategies

• Combined modeling to simulate in vivo interaction processes

These computational methods can guide antibody design to specifically target interaction interfaces .

How can PNPLA7 antibodies be optimized for studying its role in neurological conditions?

PNPLA7's relationship to neuropathy target esterase suggests important neurological functions:

Neurological Research Applications:

• Antibody selection considerations:

  • Choose antibodies that recognize neuronal PNPLA7 isoforms

  • Validate in neural tissues (Neuro-2a, primary neurons)

  • Ensure blood-brain barrier penetration for in vivo studies

• Specialized techniques:

  • Immunohistochemistry optimized for neural tissues

  • In situ proximity ligation assays to study interactions in neurons

  • Live-cell imaging with function-blocking antibodies

• Experimental models:

  • Compare PNPLA7 expression across various neuronal subtypes

  • Study changes during neurodegeneration or after neurotoxin exposure

  • Investigate PNPLA7's role in maintaining axonal integrity

Key Methodological Considerations:

• Use proper fixation methods to preserve neural tissues

• Include appropriate neuronal markers for co-localization studies

• Consider region-specific differences in PNPLA7 expression

• Control for lipid-rich environment of neural tissues affecting antibody performance