SMPDL3A Antibody

What is SMPDL3A and why is it significant for research?

SMPDL3A (Sphingomyelin Phosphodiesterase Acid Like 3A) is a protein with nucleotide phosphodiesterase activity that has garnered research interest due to its functional properties. Despite its name suggesting sphingomyelinase activity, SMPDL3A actually functions as a phosphodiesterase with activity against nucleotide diphosphate and triphosphate substrates at both acidic and neutral pH . Its significance stems from its role in macrophage function and potential involvement in the pathobiology of atherosclerosis, as it is upregulated in cholesterol-loaded macrophages . The protein is also detectable in circulating blood, suggesting potential biomarker applications . Understanding SMPDL3A function provides insights into novel anti-inflammatory mechanisms linking lipid metabolism with purinergic signaling.

What types of SMPDL3A antibodies are available for research applications?

SMPDL3A antibodies are available in various formats designed for different experimental applications. Polyclonal antibodies raised in rabbits are commonly used, with demonstrated reactivity to human SMPDL3A and predicted cross-reactivity with mouse and rat homologs . These antibodies are typically validated for multiple applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) . Commercial antibodies are often produced using recombinant protein fragments (such as amino acids 132-320 of human SMPDL3A) as immunogens . Both unconjugated primary antibodies and detection antibodies specifically designed for sandwich ELISA systems are available to researchers .

What are the recommended experimental conditions for Western blotting with SMPDL3A antibodies?

For Western blotting applications using SMPDL3A antibodies, the following methodological approach is recommended: Prepare protein samples by boiling in SDS-PAGE sample buffer containing 1% SDS, 100 mM DTT, and 60 mM Tris·HCl (pH 6.8) . Separate samples on 4-12% gradient bis-Tris gels and transfer to nitrocellulose membranes . Block membranes in PBS containing 4% (w/v) skim milk and 0.1% (v/v) Tween 20 for 1 hour at room temperature . Incubate with primary SMPDL3A antibody at a dilution of 1:500 to 1:2000 (optimal dilution should be determined empirically) . Follow with appropriate HRP-conjugated secondary antibody incubation for 1 hour . When interpreting results, note that SMPDL3A appears at approximately 51 kDa on Western blots .

How should SMPDL3A antibodies be stored and reconstituted to maintain optimal activity?

SMPDL3A antibodies are typically supplied in lyophilized form and require proper reconstitution and storage to maintain functionality. To reconstitute lyophilized antibody, add 100 μl of sterile distilled water containing 50% glycerol . After reconstitution, the antibody concentration is typically 1 mg/ml . For storage, keep reconstituted antibody at -20°C and avoid repeated freeze/thaw cycles which can compromise antibody performance . Before lyophilization, antibodies are typically formulated in a buffer containing 1% BSA and 0.02% NaN3, which helps maintain stability . Follow manufacturer guidelines for specific antibody formulations, as storage conditions may vary by supplier and antibody type.

What controls should be included when using SMPDL3A antibodies in experimental procedures?

When designing experiments using SMPDL3A antibodies, incorporate appropriate controls to ensure result validity. For Western blotting, include positive controls (tissues or cells known to express SMPDL3A, such as human macrophages) . Include negative controls such as samples from SMPDL3A-knockout models or cells with low expression levels. For loading controls, use housekeeping proteins appropriate for your sample type. In ELISA applications, establish standard curves using recombinant SMPDL3A protein at known concentrations (typically in the range of 0.79-50 ng/mL) . Include blank wells (no antigen) to determine background signal levels. When performing IHC, include isotype controls (using non-specific IgG of the same isotype as the primary antibody) and tissue sections known to be negative for SMPDL3A expression.

How can SMPDL3A antibodies be used to investigate its role in atherosclerosis and inflammatory processes?

SMPDL3A expression is upregulated in cholesterol-loaded macrophages and may play a role in atherosclerosis through a novel anti-inflammatory mechanism linking lipid metabolism with purinergic signaling . To investigate this, researchers can design experiments using SMPDL3A antibodies in several ways: Perform immunohistochemistry on atherosclerotic plaques using anti-SMPDL3A antibodies at dilutions of 1:100-1:200 to visualize protein localization within lesions . Conduct Western blotting on macrophage lysates after cholesterol loading or LXR stimulation to quantify SMPDL3A upregulation . Use ELISA to measure secreted SMPDL3A in culture supernatants from treated macrophages or in plasma from atherosclerosis models . Combine these approaches with functional assays measuring nucleotide phosphodiesterase activity to correlate SMPDL3A expression with extracellular ATP degradation and subsequent anti-inflammatory effects in the atherosclerotic microenvironment.

What methodological considerations are important when developing a sandwich ELISA for SMPDL3A detection?

Developing a robust sandwich ELISA for SMPDL3A requires careful consideration of several methodological factors. The standard assay employs a capture antibody pre-coated onto microplate wells that specifically recognizes human SMPDL3A . After sample addition and washing, add a detection antibody specific for SMPDL3A, followed by an enzyme conjugate system . For optimal results, follow this procedural sequence: (1) Coat wells with capture antibody; (2) Block non-specific binding sites; (3) Add samples or standards (0.79-50 ng/mL range); (4) Incubate for 2 hours at 37°C; (5) Add working biotin-conjugated detection antibody; (6) Incubate for 1 hour at 37°C; (7) Add working streptavidin-HRP; (8) Incubate for 1 hour at 37°C; (9) Add substrate solution and incubate 15-20 minutes at 37°C under dark conditions; (10) Add stop solution and measure optical density at 450nm . The assay typically has a detection range of 0.79-50 ng/mL with a minimum detectable dose below 0.39 ng/mL .

How can researchers address cross-reactivity concerns when working with SMPDL3A antibodies?

Cross-reactivity is an important consideration when working with SMPDL3A antibodies, particularly given the existence of related proteins like SMPDL3B. While commercial antibodies are tested for specificity, researchers should implement additional validation steps . First, conduct epitope analysis by comparing the immunogen sequence used to generate the antibody against sequences of potential cross-reactive proteins using bioinformatics tools. For polyclonal antibodies raised against recombinant fragments (like the 132-320 amino acid region of human SMPDL3A) , test antibody performance in systems with SMPDL3A knockout or knockdown. Perform pre-absorption controls by incubating the antibody with excess purified antigen before use in the intended application. When interpreting results, consider that while some antibodies are predicted to cross-react with mouse and rat SMPDL3A , experimental validation of cross-species reactivity should be performed in your specific experimental system.

What approaches can be used to troubleshoot inconsistent SMPDL3A detection in Western blotting?

When encountering inconsistent SMPDL3A detection in Western blotting, systematic troubleshooting is essential. First, verify sample preparation: SMPDL3A is a 51 kDa protein, and proper denaturation in sample buffer containing 1% SDS and 100 mM DTT is critical . Optimize antibody dilution by testing a range (1:500-1:2000) to determine the optimal concentration for your specific experimental system . Consider the blocking buffer composition; PBS with 4% skim milk and 0.1% Tween 20 is recommended . For glycosylated forms of SMPDL3A (noted in circulation) , inconsistent bands may reflect different glycosylation states; consider enzymatic deglycosylation of samples before electrophoresis. If detection remains problematic, evaluate alternative antibodies targeting different epitopes of SMPDL3A. For low abundance samples, incorporate enrichment steps such as immunoprecipitation before Western blotting or use more sensitive detection methods like chemiluminescence with extended exposure times.

How can SMPDL3A antibodies be utilized to investigate the protein's nucleotide phosphodiesterase activity?

SMPDL3A possesses unexpectedly strong nucleotide phosphodiesterase activity, particularly against nucleotide triphosphates despite its name suggesting sphingomyelinase activity . To investigate this enzymatic function, researchers can employ SMPDL3A antibodies in several experimental approaches: Use immunoprecipitation with SMPDL3A antibodies to isolate the protein from cell lysates or culture supernatants, then assess enzymatic activity against nucleotide substrates in the immunoprecipitated material. Perform immunodepletion experiments by removing SMPDL3A from macrophage secretions using specific antibodies, then measuring the remaining nucleotide phosphodiesterase activity to determine SMPDL3A's contribution to total activity . For in situ analysis, combine immunofluorescence using SMPDL3A antibodies with fluorescent nucleotide analogs to visualize enzyme-substrate interactions in cellular contexts. Develop activity assays incorporating SMPDL3A antibodies in either inhibitory or non-inhibitory configurations to distinguish between SMPDL3A-dependent and independent phosphodiesterase activities in complex biological samples.

What factors influence the linearity and recovery rates in SMPDL3A ELISA assays?

The reliability of SMPDL3A ELISA assays depends on linearity across dilutions and accurate analyte recovery. Based on validation studies, several factors influence these parameters: Sample matrix effects are significant—serum samples show recovery rates averaging 88% (range 83-107%), while cell culture media demonstrate higher recovery at 103% (range 90-106%) . Linearity varies with dilution factors: 1:2 dilutions show 82-97% recovery, 1:4 dilutions 94-98%, 1:8 dilutions 91-105%, and 1:16 dilutions 91-95% of expected values . To optimize assay performance, researchers should use the appropriate sample diluent recommended by the manufacturer. Sample preparation techniques, including any extraction or pre-processing steps, should be standardized. Standard curve preparation is critical—ensure accurate serial dilutions and use freshly prepared standards when possible. For samples with high SMPDL3A concentrations exceeding the standard curve, perform dilutions within the validated linearity range and multiply by the dilution factor to calculate the final concentration .

How does the pH-dependent activity of SMPDL3A impact antibody selection and experimental design?

SMPDL3A exhibits pH-dependent enzymatic activity, functioning at both acidic and neutral pH levels, which has important implications for antibody selection and experimental design . When selecting antibodies for functional studies, consider whether the epitope recognized might be affected by pH-induced conformational changes. For antibodies targeting regions involved in substrate binding or catalysis, binding efficiency may vary under different pH conditions. When designing experiments to assess SMPDL3A activity, incorporate buffers at various pH values (particularly acidic and neutral) to capture the full range of enzymatic function . For immunoprecipitation followed by activity assays, select antibodies that maintain binding capacity across the pH range of interest. In localization studies, consider that different cellular compartments have distinct pH environments—antibodies may perform differently when detecting SMPDL3A in acidic compartments versus neutral environments. When measuring SMPDL3A in extracellular fluids or culture media, account for the impact of environmental pH on both antigen-antibody interactions and enzymatic activity.

What approaches can be used to optimize immunohistochemistry protocols for SMPDL3A detection in tissue sections?

Optimizing immunohistochemistry (IHC) protocols for SMPDL3A detection requires attention to several technical aspects. Antigen retrieval is critical—SMPDL3A is a secreted protein that may form complexes with other molecules, potentially masking epitopes; test both heat-induced epitope retrieval (citrate buffer, pH 6.0) and enzymatic retrieval methods to determine optimal conditions. Antibody concentration should be carefully titrated; starting with the recommended dilution range (1:100-1:200) , perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background. Incubation conditions affect antibody penetration and binding; test both overnight incubation at 4°C and shorter incubations at room temperature. Detection system selection impacts sensitivity; compare chromogenic (DAB) versus fluorescent detection to determine which better visualizes SMPDL3A in your tissue type. Consider dual-staining approaches combining SMPDL3A antibodies with markers of macrophages or foam cells when investigating atherosclerotic tissues, given SMPDL3A's upregulation in cholesterol-loaded macrophages .

How can researchers quantitatively assess SMPDL3A expression levels in different experimental systems?

Quantitative assessment of SMPDL3A expression requires selecting appropriate methodologies based on the experimental system and research question. For protein-level quantification, Western blotting with densitometric analysis can be used to measure relative SMPDL3A levels between conditions, normalizing to loading controls . ELISA provides more precise quantification, with commercial kits offering a detection range of 0.79-50 ng/mL and sensitivity below 0.39 ng/mL . For tissue samples, quantitative immunohistochemistry can be performed using image analysis software to measure staining intensity and distribution. At the cellular level, flow cytometry using fluorophore-conjugated SMPDL3A antibodies enables quantification on a per-cell basis. For mRNA expression analysis, quantitative RT-PCR targeting SMPDL3A transcripts complements protein-level measurements. When comparing across different experimental systems, incorporate standardized positive controls (e.g., LXR-stimulated macrophages known to express high SMPDL3A levels) to enable cross-experimental normalization and ensure reliable quantitative comparisons.

What considerations are important when designing co-immunoprecipitation experiments to study SMPDL3A-protein interactions?

Co-immunoprecipitation (co-IP) experiments to investigate SMPDL3A-protein interactions require careful consideration of several factors. Antibody selection is critical—choose antibodies that recognize epitopes not involved in potential protein-protein interactions; the antibody targeting the recombinant fragment corresponding to amino acids 132-320 may be suitable if interaction domains lie outside this region . Lysis buffer composition significantly impacts experimental success; for studying SMPDL3A interactions, consider buffers that preserve protein-protein interactions while efficiently extracting SMPDL3A (typically containing 1% non-ionic detergents like NP-40 or Triton X-100). Pre-clearing lysates with appropriate control beads reduces non-specific binding. Washing stringency affects the detection of weak versus strong interactions; optimize salt concentration and detergent levels in wash buffers based on the expected strength of the interactions being studied. For secreted SMPDL3A , consider performing co-IP from concentrated culture supernatants in addition to cell lysates. When analyzing co-IP results, include appropriate controls: IgG isotype control, input sample (pre-IP lysate), and reciprocal IP (using antibodies against suspected interacting partners to pull down SMPDL3A).

How do post-translational modifications of SMPDL3A affect antibody recognition and functional studies?

SMPDL3A undergoes post-translational modifications, including N-glycosylation, which can significantly impact antibody recognition and functional analysis . When interpreting Western blot results, researchers should expect potential band shifts or multiple bands representing differently modified forms of SMPDL3A; the observed molecular weight of 51 kDa may vary based on modification state . For comprehensive characterization, compare results from samples treated with glycosidases to remove N-glycans. When selecting antibodies for specific applications, consider whether they target regions containing modification sites—antibodies recognizing peptide sequences may perform differently than those recognizing conformational epitopes potentially affected by glycosylation. For ELISA assays, evaluate whether the capture and detection antibodies recognize native glycosylated SMPDL3A in biological samples with the same efficiency as recombinant standards, which may have different modification patterns . In functional studies, assess whether enzymatic activity correlates with specific modified forms by combining immunoprecipitation of different SMPDL3A forms (separated by size or using modification-specific antibodies) with activity assays.

What experimental approaches can address the distinction between SMPDL3A and classical acid sphingomyelinase when studying phosphodiesterase activities?

Despite its name suggesting sphingomyelinase-like activity, SMPDL3A actually functions as a nucleotide phosphodiesterase without detectable sphingomyelin phosphodiesterase activity . To experimentally distinguish between SMPDL3A and classical acid sphingomyelinase (ASM), researchers should implement several approaches: Substrate specificity assays using sphingomyelin, nucleotide di/triphosphates, and other potential substrates allow biochemical differentiation; SMPDL3A shows activity against ATP and other nucleotide triphosphates but not sphingomyelin . Selective antibody-mediated depletion from samples followed by activity assays allows determination of which protein contributes to observed activities. pH-dependent activity profiles provide further distinction—while both enzymes function at acidic pH, specific pH optima differ and SMPDL3A maintains significant activity at neutral pH unlike classical ASM . Inhibitor response profiles using known ASM inhibitors help differentiate the enzymes; test whether SMPDL3A activity is affected by these compounds. Expression pattern analysis in response to stimuli such as LXR agonists or cholesterol loading, which specifically upregulate SMPDL3A in human macrophages, provides additional differentiation criteria .

How can researchers interpret SMPDL3A levels in the context of macrophage cholesterol loading and atherosclerosis studies?

SMPDL3A is upregulated in cholesterol-loaded macrophages and potentially plays a role in atherosclerosis, requiring careful interpretation of expression data in this context . When analyzing SMPDL3A levels in atherosclerosis studies, consider several key factors: Temporal dynamics—assess whether SMPDL3A upregulation precedes, coincides with, or follows foam cell formation to establish causality relationships. Localization patterns—determine whether SMPDL3A is primarily cell-associated or secreted into the extracellular environment, as its secretion may affect local nucleotide concentrations and subsequent inflammatory signaling . Correlation with inflammatory markers—examine whether SMPDL3A levels inversely correlate with pro-inflammatory cytokine expression or macrophage activation state, consistent with its proposed anti-inflammatory role. Functional consequences—measure nucleotide phosphodiesterase activity in parallel with SMPDL3A levels to confirm that expression changes translate to altered enzymatic function. Species differences—note that while SMPDL3A antibodies may cross-react with mouse and rat homologs , regulation of SMPDL3A may differ between species, potentially affecting translational relevance of animal model findings.

What approaches can researchers use to validate knockdown or overexpression of SMPDL3A in functional studies?

Validation of SMPDL3A knockdown or overexpression is essential for establishing causality in functional studies. For protein-level validation, Western blotting using SMPDL3A antibodies at 1:500-1:2000 dilution provides semi-quantitative assessment of expression changes . Quantitative ELISA offers more precise measurement, with sensitivity below 0.39 ng/mL for detecting changes in both cellular and secreted SMPDL3A levels . At the mRNA level, quantitative RT-PCR targeting SMPDL3A transcripts complements protein assessment. Functional validation through nucleotide phosphodiesterase activity assays confirms that expression changes translate to altered enzymatic function . For overexpression studies, researchers can utilize constructs like the tetracycline-inducible C-terminal His6-tagged SMPDL3A expression system (from plasmid pcDNA 4/TO-SMPDL3A6His) . This allows for controlled expression and easy detection via either SMPDL3A or His-tag antibodies. For knockdown validation, assess potential compensatory upregulation of related enzymes like SMPDL3B. When designing rescue experiments, use expression constructs containing silent mutations that render the mRNA resistant to siRNA/shRNA while maintaining the wild-type protein sequence.

How can researchers use SMPDL3A antibodies to investigate its potential role in diseases beyond atherosclerosis?

While SMPDL3A has been studied in the context of atherosclerosis, its nucleotide phosphodiesterase activity and expression patterns suggest potential roles in other diseases that researchers can investigate using specific antibodies . For immunological disorders, use SMPDL3A antibodies in immunohistochemistry (1:100-1:200 dilution) to examine expression in tissues from inflammatory disease models, focusing on macrophage-rich regions. In metabolic diseases, quantify SMPDL3A in serum/plasma using ELISA (detection range 0.79-50 ng/mL) to determine whether levels correlate with disease progression or response to therapy. For cancer research, examine SMPDL3A expression in tumor-associated macrophages versus normal tissue macrophages, as altered purinergic signaling affects tumor microenvironments. In kidney disease research, investigate SMPDL3A in relation to SMPDL3B, which has established roles in kidney function. For parasitic infections like polycystic echinococcosis (associated with SMPDL3A in GeneCards data) , examine whether SMPDL3A is involved in host-pathogen interactions. When designing such studies, incorporate tissue microarrays or multiple sample cohorts to efficiently screen SMPDL3A expression across diverse pathological states.

What are the key differences between SMPDL3A and its paralog SMPDL3B in terms of antibody selection and experimental applications?

SMPDL3A and its paralog SMPDL3B share structural similarities but have distinct functions, necessitating careful antibody selection and experimental design when studying either protein . For antibody selection, prioritize antibodies validated for specificity against the target paralog; verify the immunogen sequence used to generate the antibody and confirm it does not have high homology with regions in the other paralog. When designing experiments, consider the differing tissue expression patterns—while SMPDL3A is upregulated in cholesterol-loaded macrophages , SMPDL3B may have different cellular distribution and regulation. For functional studies, note that despite structural similarities, the proteins may have distinct substrate preferences and enzymatic properties; design activity assays accordingly. When interpreting results, be aware that commercial antibodies predicted to cross-react with mouse and rat SMPDL3A may have different cross-reactivity profiles for SMPDL3B. When studying both paralogs simultaneously (e.g., to assess potential compensatory mechanisms), design multiplexed detection methods using antibodies with confirmed paralog specificity and compatible detection systems.

How can precision and reproducibility be maximized in SMPDL3A detection and quantification across different laboratories?

Maximizing precision and reproducibility in SMPDL3A research across laboratories requires standardization of several critical factors. Antibody selection and validation should follow consistent protocols—the same antibody clones, formats, and lot numbers ideally should be used across studies, with validation data shared between laboratories. For ELISA-based quantification, established commercial kits demonstrate intra-plate precision with CV<10% and inter-plate precision with CV<15% ; laboratories should implement similar quality control measures. Sample collection and processing protocols should be standardized, particularly for serum samples where recovery rates average 88% (range 83-107%) . Data normalization approaches should be consistent—whether normalizing Western blot densitometry to specific loading controls or ELISA values to standard reference materials. Round-robin testing between laboratories using the same samples can identify systematic biases. Detailed reporting of all methodological parameters in publications facilitates reproduction, including antibody dilutions (e.g., 1:500-1:2000 for Western blotting, 1:100-1:200 for IHC) , incubation conditions, detection methods, and data analysis approaches.

What emerging technologies might enhance the study of SMPDL3A expression and function beyond traditional antibody applications?

Beyond traditional antibody-based methods, several emerging technologies hold promise for advancing SMPDL3A research. CRISPR-Cas9 gene editing enables generation of endogenously tagged SMPDL3A (e.g., with fluorescent proteins or affinity tags), allowing real-time visualization or purification without relying on antibodies. Proximity labeling approaches like BioID or APEX can map SMPDL3A's protein interaction network by identifying proteins in close proximity in living cells. Mass spectrometry-based proteomics offers antibody-independent quantification of SMPDL3A and its post-translational modifications, with parallel reaction monitoring providing highly specific quantification. Single-cell technologies including single-cell RNA-seq and mass cytometry can reveal cell-specific expression patterns of SMPDL3A in heterogeneous populations like atherosclerotic plaques. Organ-on-chip systems incorporating macrophages can model SMPDL3A regulation in a physiologically relevant microenvironment. For functional studies, activity-based protein profiling using modified nucleotide substrates could selectively label active SMPDL3A in complex samples. These approaches complement rather than replace antibody-based methods, potentially overcoming limitations related to antibody specificity and providing additional layers of information about SMPDL3A biology.

How might the development of monoclonal antibodies against different SMPDL3A epitopes advance functional studies of the protein?

Development of monoclonal antibodies against distinct epitopes of SMPDL3A would significantly enhance functional studies by providing more specific tools for different applications. Antibodies targeting the catalytic domain could potentially modulate enzymatic activity, serving as functional probes to investigate SMPDL3A's nucleotide phosphodiesterase activity . Conformation-specific antibodies recognizing either active or inactive states would enable assessment of SMPDL3A's activation status in different cellular contexts. Epitope-specific antibodies that distinguish between different post-translationally modified forms (particularly glycosylated variants) would allow investigation of modification-specific functions . Domain-specific antibodies could help map protein-protein interaction interfaces by determining which regions are accessible or masked when SMPDL3A forms complexes. Antibodies suitable for super-resolution microscopy would enable detailed localization studies at the subcellular level. Combined with the existing polyclonal antibodies that recognize amino acids 132-320 , a panel of epitope-specific monoclonal antibodies would create a comprehensive toolkit for SMPDL3A research, enabling more precise manipulation and analysis of this multifunctional protein in diverse experimental settings.