Patatin Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What is patatin and why are patatin antibodies important in research?
Patatin is a family of glycoproteins found primarily in potato tubers (Solanum tuberosum), constituting approximately 40-60% of the soluble protein content . These proteins consist of approximately 366 amino acids with an isoelectric point of 4.9 and molecular weights ranging from 40-45 kDa, though they commonly exist as 80 kDa dimers without disulfide bridges . Patatin antibodies are essential research tools for several reasons: they enable precise quantification of patatin levels in plant tissues, facilitate immunolocalization studies to determine subcellular distribution, and support investigations into patatin's multifunctional roles in both plant physiology and human health applications . Beyond their use in plant biology, patatin antibodies have become increasingly valuable for studying homologous patatin-like phospholipase domain-containing proteins (PNPLAs) in animals and microorganisms, which share structural and functional characteristics with plant patatins .
How should I select the appropriate patatin antibody for my research application?
Selection of the appropriate patatin antibody depends on multiple experimental factors. First, consider the specific patatin isoform of interest—potato patatins have at least 36 known isoforms, and antibodies may exhibit differential reactivity . Polyclonal antibodies, like the commercially available rabbit anti-patatin, offer broad recognition of multiple epitopes, which can be advantageous for detection but may introduce cross-reactivity issues . When choosing an antibody, evaluate the documented reactivity spectrum, the host species (commonly rabbit), and the specific application validations (Western blot, immunoprecipitation, immunolocalization) . For Western blot applications, antibodies validated at dilutions of approximately 1:2000 typically provide good signal-to-noise ratios . Always review the antibody's immunogen information—for instance, some commercial antibodies are developed against KLH-conjugated synthetic peptides derived from C-terminal regions common to multiple patatin isoforms . Additionally, consider whether your experimental design requires detection of native or denatured forms of patatin, as this will influence antibody selection.
What protocols work best for sample preparation when using patatin antibodies?
Effective sample preparation is critical for successful patatin antibody applications. For protein extraction from potato tissue, a buffer containing 20 mM Tris (pH 8.5), 10 mM thiourea, 10 mM CaCl₂, 5 mM DTT, 1 mM PMSF, and 1% PVPP has been documented to yield good results . This combination preserves patatin integrity while minimizing interference from phenolic compounds and other plant components. For Western blot applications, separating approximately 75 μg of total protein on 4-20% SDS-PAGE gels followed by transfer to PVDF membranes (1.5 hours) provides optimal resolution of patatin bands . Blocking with 5% milk powder in T-TBS for 1 hour at room temperature helps minimize non-specific binding. For immunolocalization studies, fixation protocols must be carefully optimized as overfixation may mask epitopes; a 1:100 dilution of primary antibody has been found effective for such applications . When working with patatin-like proteins from non-plant sources, such as microbial or human samples, extraction buffers may need to be modified to account for different cellular compositions and protein solubility characteristics.
How can I validate the specificity of my patatin antibody in experimental systems?
Validating antibody specificity is a critical step for ensuring reliable experimental results. Begin with a multi-pronged approach including positive and negative controls. For positive controls, use purified patatin protein or well-characterized potato extracts with known patatin content . Negative controls should include non-potato plant tissues and knockout or knockdown systems when available. Western blot analysis should demonstrate bands at the expected molecular weight (40-42 kDa for monomeric patatin) . Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide before application to samples, can confirm binding specificity—signal abolishment indicates specific binding. For cross-reactivity assessment, test the antibody against proteins with structural similarity to patatin, such as other lipases or patatin-like domain proteins from different species . If studying patatin-like proteins in non-plant systems, like the PNPLA family in humans, verification can include heterologous expression systems where target proteins are tagged (e.g., FLAG-tagged PNPLA3) and detected with both the patatin antibody and an anti-tag antibody to confirm co-localization . Finally, immunoprecipitation followed by mass spectrometry can provide definitive identification of the proteins being recognized by your antibody.
What are the optimal conditions for extracting and preserving patatin for antibody studies?
Patatin extraction and preservation require careful attention to protein stability and enzymatic activity. For total protein extraction from potato tubers, a buffer containing 20 mM Tris (pH 8.5), 10 mM thiourea, 10 mM CaCl₂, 5 mM DTT, 1 mM PMSF, and 1% PVPP effectively solubilizes patatin while inhibiting proteases and preventing oxidation . The inclusion of PVPP is particularly important for removing phenolic compounds that can interfere with downstream applications. Temperature control during extraction is critical—maintain samples at 4°C throughout the process to prevent degradation. For long-term storage of extracted proteins, flash freezing aliquots in liquid nitrogen followed by storage at -80°C preserves immunoreactivity better than storage at -20°C. If studying patatin enzymatic activity, glycerol (10-20%) can be added to storage buffers to maintain functional conformation. When working with tissue samples for immunohistochemistry, fixation in 4% paraformaldehyde for 24 hours followed by careful washing and dehydration provides good epitope preservation. For immunolocalization studies in plant tissues, cryosectioning often preserves antigenicity better than paraffin embedding, which can mask epitopes through heat and chemical modification. If long-term storage of antibodies themselves is required, lyophilization followed by reconstitution in sterile water has been shown to maintain reactivity, with recommended storage of reconstituted antibody in aliquots at -20°C to avoid freeze-thaw cycles .
How can I optimize Western blot protocols specifically for patatin detection?
Optimizing Western blot protocols for patatin detection requires attention to several key parameters. First, sample preparation is critical—aim for 75 μg of total protein from potato extracts for clear detection . Gradient gels (4-20% SDS-PAGE) provide optimal separation of patatin from other proteins . For transfer, PVDF membranes generally yield better results than nitrocellulose for patatin proteins, with a recommended transfer time of 1.5 hours . Blocking with 5% milk powder in T-TBS for 1 hour at room temperature effectively minimizes background without compromising specific binding . Primary antibody concentration should be optimized through titration experiments, with 1:2000 dilution often providing good results for commercial anti-patatin antibodies . Overnight incubation at 4°C with gentle agitation improves binding efficiency compared to shorter incubations. For detection, HRP-conjugated secondary antibodies at 1:10,000 dilution coupled with enhanced chemiluminescence (ECL) provide sensitive detection with minimal background . Exposure times of approximately 30 seconds are typically sufficient, though this may vary with sample concentration and antibody batch . When working with multiple patatin isoforms, longer separation times and higher percentage gels may be necessary to resolve closely related variants. For challenging samples, addition of 0.1% SDS to the antibody dilution buffer can reduce non-specific binding while maintaining specific interactions with patatin.
How can immunohistochemistry with patatin antibodies enhance understanding of subcellular localization?
Immunohistochemistry (IHC) with patatin antibodies offers significant insights into the subcellular distribution and functional contexts of patatin proteins. To achieve optimal results, tissue fixation methods must be carefully selected—paraformaldehyde fixation (4%, 24 hours) preserves tissue architecture while maintaining epitope accessibility. For potato tissues, cryosectioning at 10-15 μm thickness often provides better antigen preservation than paraffin embedding. Antigen retrieval techniques, such as citrate buffer treatment (pH 6.0, 95°C for 20 minutes), may be necessary to unmask epitopes obscured during fixation. A dilution of 1:100 for primary anti-patatin antibodies has proven effective for IHC applications . For fluorescence detection, secondary antibodies conjugated to bright, photostable fluorophores like Alexa Fluor dyes enable precise localization studies when combined with confocal microscopy. This approach has revealed that patatin is predominantly localized within vacuoles of parenchyma cells in potato tubers , but can also be detected at lower levels in other cellular compartments during certain developmental stages. Co-localization studies using antibodies against organelle markers (e.g., tonoplast proteins for vacuoles) in combination with patatin antibodies can provide definitive evidence of subcellular distribution. For human or animal tissues expressing patatin-like proteins, such as PNPLA2/PEDFR, antibodies specifically developed against these homologs allow for comparative localization studies across species . Importantly, immunohistochemical approaches can track dynamic changes in patatin distribution during development, stress responses, or disease progression, offering temporal insights not available through biochemical approaches alone.
What approaches are recommended for studying interactions between patatin and other proteins?
Studying protein-protein interactions involving patatin requires specialized approaches due to patatin's enzymatic activities and structural characteristics. Co-immunoprecipitation (Co-IP) using anti-patatin antibodies followed by mass spectrometry represents a powerful unbiased approach for identifying interaction partners. For this technique, mild lysis conditions using buffers containing 0.5% NP-40 or 1% Triton X-100 help preserve protein complexes. Cross-linking reagents such as formaldehyde or DSP (dithiobis[succinimidyl propionate]) can stabilize transient interactions prior to immunoprecipitation. Proximity-based labeling methods, including BioID or APEX2, offer alternatives for capturing interactions in their native cellular environment—fusion of these enzymes to patatin allows biotinylation of nearby proteins, which can then be purified and identified. Yeast two-hybrid screening provides another approach, though care must be taken to account for patatin's lipase activity, which may affect yeast viability. For patatin-like proteins in mammals, such as the PNPLA family members, split-luciferase complementation assays in mammalian cells offer sensitive detection of protein interactions in living cells. Importantly, validation of identified interactions should employ multiple orthogonal methods, including reverse Co-IP, where antibodies against the putative interacting partner are used to precipitate complexes and patatin is detected by Western blot. Functional validation through mutagenesis of interaction domains provides additional evidence of biological relevance. When studying interactions with lipid substrates or membranes, techniques such as liposome binding assays or lipid overlay assays can complement protein-protein interaction studies to develop a comprehensive understanding of patatin's functional interactions.
How can researchers study the role of patatin-like proteins in human diseases using antibody-based approaches?
Investigating patatin-like proteins in human disease contexts requires specialized antibody-based approaches tailored to clinical samples. Immunohistochemistry and immunofluorescence microscopy using qualified antibodies against human patatin-like proteins, such as PNPLA2 (ATGL) or PNPLA3, can reveal changes in expression patterns and subcellular localization associated with disease progression . For example, PNPLA3 has been studied in non-alcoholic fatty liver disease (NAFLD) patients using immunohistochemistry to correlate protein levels with disease severity and genetic variants . When working with clinical specimens, proper antibody validation is critical—this should include genetic controls (tissues from patients with different PNPLA genotypes) and peptide competition assays to ensure specificity . Multiplexed immunofluorescence, where antibodies against patatin-like proteins are combined with markers of cell type, organelles, or disease state, can provide contextual information about expression patterns. For quantitative assessment, tissue microarrays containing samples from multiple patients enable high-throughput analysis of protein expression across disease stages. In circulation-based studies, immunoassays like ELISA or multiplexed approaches such as Luminex can measure soluble forms of patatin-like proteins as potential biomarkers. When correlating with genetic variants, such as the PNPLA3 rs738409 polymorphism, integrated approaches combining genotyping, protein quantification, and functional assays provide comprehensive insights into genotype-phenotype relationships . For mechanistic studies, patient-derived cells expressing patatin-like proteins can be analyzed using proximity ligation assays to visualize and quantify protein interactions in situ, offering insights into how disease-associated variants affect protein function through altered interaction networks.
What are common causes of non-specific binding when using patatin antibodies and how can they be addressed?
Non-specific binding represents one of the most common challenges when working with patatin antibodies. Several factors can contribute to this issue: First, high homology between patatin isoforms (potato contains at least 36 known isoforms) can lead to cross-reactivity even with well-designed antibodies . Additionally, patatin's relatively high abundance in potato tissues (40-60% of soluble protein) can result in oversaturation signals and background . To address these issues, implement a multi-faceted optimization strategy: Increase blocking stringency by using 5% BSA instead of milk powder, or consider specialized blocking reagents containing non-mammalian proteins for reduced background . Diluting primary antibodies further than the manufacturer's recommendation (e.g., 1:5000 instead of 1:2000 for Western blots) can reduce non-specific interactions while maintaining specific signals . Including competitive blocking agents, such as non-immune serum from the same species as the secondary antibody, can minimize secondary antibody cross-reactivity. For particularly challenging samples, pre-adsorption of the primary antibody with non-target tissues (e.g., non-potato plant extracts when studying potato patatin) can remove antibodies with cross-reactivity. When working with tissues containing high levels of endogenous peroxidases or phosphatases, include appropriate quenching steps (3% H₂O₂ for peroxidases) before antibody application. Finally, increasing the number and duration of wash steps (six 5-minute washes in TBS-T) significantly reduces background without compromising specific signals . For immunohistochemistry applications, tissue-specific autofluorescence can be minimized using Sudan Black B treatment or spectral unmixing during image acquisition.
How can researchers address batch-to-batch variability in patatin antibody performance?
Batch-to-batch variability in antibody performance presents significant challenges for longitudinal studies and data reproducibility. To address this issue, implement a comprehensive validation strategy for each new antibody lot: First, establish a reference standard—maintain aliquots of a well-characterized positive control sample (e.g., standardized potato extract) and compare new antibody lots against this benchmark using consistent Western blot protocols . Document key performance metrics including signal-to-noise ratio, detection threshold, and EC50 values for quantitative comparisons between batches. Consider preparing an antibody validation panel consisting of samples with known patatin content spanning the dynamic range of detection. For critical applications, purchase larger antibody lots that can support the entire project duration, with proper aliquoting and storage at -80°C to maintain stability. When antibody performance drifts significantly between batches, normalization strategies such as calibration curves using purified patatin standards can help standardize results. For absolute quantification requirements, consider developing sandwich ELISA assays using two different anti-patatin antibodies recognizing distinct epitopes, which can provide more consistent performance across batches. Maintain detailed records of antibody lot numbers, validation data, and experimental conditions to facilitate troubleshooting when performance issues arise. When transitioning between antibody lots is unavoidable, conduct side-by-side comparisons using both lots on identical samples to establish conversion factors for data normalization. Finally, consider polyclonal antibody pooling strategies, where small amounts of the previous validated lot are mixed with the new lot to minimize sudden performance shifts.
What approaches can resolve detection issues when working with patatin-like proteins from different species?
Detection of patatin-like proteins across species presents unique challenges due to sequence divergence and structural variations. When antibodies raised against potato patatin fail to detect homologs in other species, several approaches can improve cross-species detection: First, perform sequence alignment analysis of the target protein with potato patatin to identify regions of highest conservation, which may serve as common epitopes recognized by anti-patatin antibodies . For patatin-like proteins in bacteria like Mycobacterium tuberculosis or human PNPLA family members, focus on the conserved patatin domain rather than full-length protein detection . Consider using antibodies developed against synthetic peptides representing the most conserved regions of the patatin domain. When commercial antibodies are insufficient, custom antibody development using recombinant proteins expressing only the conserved patatin domain can improve cross-species detection. For enhanced sensitivity in challenging samples, employ signal amplification methods such as tyramide signal amplification (TSA) or polymer-based detection systems, which can detect low abundance proteins while maintaining specificity. Pre-enrichment techniques, including immunoprecipitation or affinity purification before Western blotting, can concentrate target proteins below the detection threshold in direct assays. For advanced research applications, structural prediction tools like AlphaFold2 can identify structurally conserved epitopes despite sequence divergence, guiding epitope-specific antibody selection . When validating detection in new species, recombinant expression of the target protein with an epitope tag (e.g., FLAG) allows confirmation of antibody binding to the correct protein through dual detection with anti-tag and anti-patatin antibodies . Finally, mass spectrometry-based targeted proteomics approaches can complement antibody-based detection for definitive identification of patatin-like proteins across diverse species.
How are patatin antibodies being utilized to study the role of patatin-like domains in microbial pathogens?
Patatin antibodies are becoming valuable tools for investigating patatin-like proteins in microbial pathogens, particularly in Mycobacterium tuberculosis, which contains an expanded family of these proteins compared to non-pathogenic mycobacteria . Researchers are utilizing structure prediction-based approaches, combined with antibody-based detection methods, to elucidate the functions of these proteins in pathogenesis . Anti-patatin domain antibodies enable immunolocalization studies revealing that these proteins often associate with bacterial cell membranes, consistent with their predicted lipase activities . Recent studies have employed immunoprecipitation coupled with mass spectrometry to identify interaction partners of patatin-like proteins in mycobacteria, uncovering connections to lipid metabolism pathways critical for virulence. Importantly, antibodies recognizing conserved patatin domains have revealed differential expression patterns of these proteins during various stages of infection and under different stress conditions, suggesting specific roles in pathogen adaptation. Cross-linking immunoprecipitation studies using custom patatin domain antibodies have identified interactions with host cell factors during infection, providing insights into host-pathogen dynamics. The development of conformation-specific antibodies that distinguish between active and inactive states of patatin-like proteins in pathogens is an emerging approach for studying their regulatory mechanisms. These antibody-based studies are complemented by structural analyses using AlphaFold2 predictions, revealing diverse regulatory domains within the patatin family that likely contribute to their specialized functions in pathogenesis . As this field advances, antibodies recognizing specific patatin-like protein isoforms in pathogens may become valuable tools for diagnostic applications and therapeutic target validation.
What novel approaches are being developed to study post-translational modifications of patatin using antibody-based methods?
Investigation of post-translational modifications (PTMs) in patatin and patatin-like proteins represents a frontier in understanding their regulation and function. Researchers are developing modification-specific antibodies that selectively recognize phosphorylated, glycosylated, or lipidated forms of patatin. For phosphorylation studies, antibodies against phospho-serine, phospho-threonine, and phospho-tyrosine can be used in combination with anti-patatin antibodies in sequential immunoprecipitation approaches to enrich modified forms. Mass spectrometry following immunoprecipitation with anti-patatin antibodies has identified several previously unknown modification sites, including phosphorylation events that appear to regulate lipase activity. For site-specific PTM detection, custom antibodies raised against synthetic peptides containing specific modified residues provide powerful tools for tracking regulatory modifications. Proximity ligation assays combining anti-patatin antibodies with PTM-specific antibodies enable in situ visualization of modified populations, revealing subcellular compartmentalization of differently modified patatin pools. For human patatin-like proteins such as PNPLA family members, studies are exploring how disease-associated variants alter modification patterns . Quantitative approaches including ELISA and Western blotting with modification-specific antibodies are being employed to determine how environmental factors and disease states impact modification levels. Advanced multiplexed imaging methods using differently labeled antibodies against various PTMs on patatin are revealing complex modification patterns that correlate with functional states. Top-down proteomics approaches following immunoaffinity enrichment with anti-patatin antibodies are providing comprehensive PTM landscapes that capture complex modification patterns across different physiological conditions. These emerging methodologies are transforming our understanding of how modifications regulate patatin's multifunctional properties across different biological contexts.
How can researchers leverage patatin antibodies to investigate evolutionary relationships between plant and animal patatin-like proteins?
Leveraging patatin antibodies for evolutionary studies of patatin-like proteins across kingdoms represents an innovative research direction with significant implications for understanding protein function evolution. Researchers are developing epitope-mapping approaches using a panel of monoclonal antibodies targeting different regions of the patatin domain to identify structurally conserved motifs between plant patatins and animal patatin-like phospholipase domain-containing proteins (PNPLAs) . Comparative immunoprecipitation studies using antibodies that recognize conserved epitopes can isolate functionally related proteins across species, enabling proteomic comparisons of interaction networks. For challenging cross-species detection, researchers are employing degenerate epitope antibodies designed to recognize structurally conserved regions despite sequence divergence. Importantly, immunohistochemical studies using these conservation-focused antibodies are revealing similar subcellular localization patterns of patatin-like proteins across evolutionarily distant organisms, suggesting functional conservation . Developmental studies tracking patatin-like protein expression during embryogenesis in various model organisms using cross-reactive antibodies are uncovering temporal parallels in expression patterns across species. Antibody-based activity assays measuring lipase function of immunoprecipitated patatin-like proteins from different species allow direct functional comparisons. Structural immunology approaches, using conformation-specific antibodies that distinguish between active and inactive states, are revealing evolutionary conservation of regulatory mechanisms. For genomic studies, chromatin immunoprecipitation using antibodies against transcription factors that regulate patatin-like protein expression across species is identifying conserved regulatory elements. These multidisciplinary approaches combining antibody-based detection with evolutionary bioinformatics are constructing a comprehensive understanding of how this ancient protein domain has been repurposed throughout evolution while maintaining core structural and functional properties.
