DMP9 Antibody

What is DPP9 and what cellular functions does it serve?

DPP9 (Dipeptidyl peptidase 9) is a serine protease that cleaves N-terminal dipeptides from proteins having a Proline or Alanine residue at position 2. It serves critical cellular functions, particularly as a key inhibitor of caspase-1-dependent monocyte and macrophage pyroptosis in resting cells by preventing activation of NLRP1 and CARD8 inflammasomes. DPP9 sequesters the cleaved C-terminal part of NLRP1 and CARD8 in a ternary complex, thereby preventing their oligomerization and activation. Interestingly, while its dipeptidyl peptidase activity is required to suppress NLRP1 and CARD8, neither of these proteins appears to be direct substrates of DPP9, suggesting the existence of other substrate(s) required for NLRP1 and CARD8 inhibition .

What are the optimal applications for DPP9 antibodies in research?

DPP9 antibodies are optimally suited for several research applications, with Western blot being one of the most validated methods. For Western blot applications, researchers can detect DPP9 at approximately 100-101 kDa in human liver tissue samples using 1 μg/mL of antibody concentration. Additionally, DPP9 antibodies work effectively in immunofluorescence studies, particularly in detecting cytoplasmic expression in cell lines such as HeLa human cervical epithelial carcinoma cells at 10 μg/mL concentration. The antibodies are also suitable for Simple Western assays and immunoprecipitation studies for protein interaction investigations .

What is the cellular localization pattern of DPP9 and how can it be visualized?

DPP9 predominantly localizes to the cytoplasm of cells. This localization pattern can be effectively visualized using immunofluorescence techniques. For example, in HeLa human cervical epithelial carcinoma cell lines, DPP9 can be detected using specific monoclonal antibodies (such as MAB5419 at 10 μg/mL) followed by fluorophore-conjugated secondary antibodies, such as NorthernLights 557-conjugated Anti-Mouse IgG. Counterstaining with DAPI helps to visualize nuclei, providing clear contrast to the cytoplasmic DPP9 staining. The specific cytoplasmic localization pattern is consistent with DPP9's role in inflammasome regulation and protein processing within the cytosolic compartment .

What are the optimal conditions for detecting DPP9 by Western blot?

For optimal detection of DPP9 by Western blot, the following conditions have been empirically determined to be effective: Use PVDF membrane and probe with 1 μg/mL of Anti-Human DPP9 Monoclonal Antibody followed by HRP-conjugated secondary antibody. The experiment should be conducted under reducing conditions using appropriate immunoblot buffer systems (such as Immunoblot Buffer Group 1). Under these conditions, DPP9 will be detected as a specific band at approximately 100 kDa in human liver tissue lysates. For Simple Western assays, load human liver tissue lysates at 0.5 mg/mL and use 10 μg/mL of Anti-Human DPP9 antibody. Be aware that non-specific interaction with high molecular weight standards may occur, so proper controls are essential .

How should researchers troubleshoot non-specific binding when using DPP9 antibodies?

When troubleshooting non-specific binding with DPP9 antibodies, researchers should implement a systematic approach. First, optimize antibody concentration by performing a titration series (typically between 0.1-10 μg/mL) to identify the minimal concentration that yields specific signal. Second, extend blocking times using 5% non-fat dry milk or BSA in TBST to reduce background signal. Third, include additional washing steps with increased stringency (0.1-0.3% Tween-20 in TBS). Fourth, validate specificity using positive and negative control samples, including DPP9-knockout or DPP9-knockdown cells. For Simple Western applications specifically, be aware that non-specific interaction with high molecular weight standards (such as the 230 kDa standard) has been reported with some DPP9 antibodies. Finally, consider pre-adsorption of antibodies with recombinant target antigens to confirm specificity of observed signals .

What immunogen characteristics should be considered when selecting a DPP9 antibody?

When selecting a DPP9 antibody, researchers should carefully evaluate the immunogen characteristics to ensure specificity and appropriate epitope recognition. Key considerations include: (1) Immunogen sequence location - antibodies targeting different regions of DPP9 may have varying accessibility to epitopes depending on protein folding and complex formation. For example, antibodies targeting epitopes within the aa 750-850 region of human DPP9 have demonstrated efficacy for immunoprecipitation studies. (2) Species cross-reactivity - confirm whether the antibody recognizes conserved epitopes across species if cross-species studies are planned. (3) Immunogen production method - recombinant protein fragments versus synthetic peptides may yield antibodies with different characteristics. (4) Post-translational modifications - consider whether the epitope region contains potential modification sites that might affect antibody binding. (5) Validation in the specific application of interest - prioritize antibodies with documented performance in your intended application using relevant biological samples .

How can DPP9 antibodies be used to investigate inflammasome regulation?

DPP9 antibodies can be powerful tools for investigating inflammasome regulation through several methodological approaches. First, co-immunoprecipitation studies using DPP9 antibodies can isolate protein complexes containing DPP9, NLRP1, and CARD8, enabling the characterization of the ternary complex that prevents inflammasome activation. Second, comparative immunofluorescence or immunohistochemistry studies in resting versus activated cells can reveal changes in DPP9 localization and association with inflammasome components. Third, proximity ligation assays using DPP9 antibodies paired with antibodies against NLRP1 or CARD8 can visualize direct interactions and how these change during inflammasome activation. Fourth, combining DPP9 antibodies with enzymatic activity assays can help determine how the dipeptidyl peptidase activity correlates with inflammasome regulation. Finally, time-course immunoblotting after inflammasome activation can track DPP9's degradation or dissociation from the inflammasome complex, providing insights into the dynamics of this regulatory system .

What strategies can be employed to study DPP9 substrate specificity using antibody-based approaches?

Studying DPP9 substrate specificity using antibody-based approaches requires sophisticated experimental designs. One effective strategy involves using DPP9 antibodies for immunoprecipitation followed by mass spectrometry (IP-MS) to identify proteins that interact with DPP9 under various cellular conditions. Researchers can also employ in situ proximity labeling techniques where DPP9 antibodies are used to validate the proximity of DPP9 to putative substrates identified through BioID or APEX approaches. Another approach involves comparing the N-terminal peptidome in cells with and without DPP9 inhibition or knockdown, using antibodies to confirm DPP9 status. Additionally, researchers can develop substrate-trapping mutants of DPP9 that bind but don't cleave substrates, then use DPP9 antibodies to immunoprecipitate these complexes. Finally, competitive binding assays using labeled potential substrates with immobilized DPP9 (captured by antibodies) can help determine binding affinities and substrate preferences, providing insights into the enigmatic relationship between DPP9's enzymatic activity and its role in inflammasome regulation .

How does DPP9 antibody detection vary across different tissue and cell types?

DPP9 antibody detection exhibits notable variations across different tissue and cell types, reflecting the differential expression and regulation of DPP9. In human liver tissue, DPP9 is readily detected by Western blot as a prominent band at approximately 100-101 kDa, indicating substantial expression in hepatic cells. In HeLa cervical carcinoma cells, immunofluorescence reveals a diffuse cytoplasmic distribution pattern, though the intensity may vary based on cell cycle or metabolic state. When comparing detection across immune cells, researchers have observed that monocytes and macrophages show particularly strong DPP9 expression, consistent with its critical role in regulating pyroptosis in these cell types. Importantly, expression levels may change during cellular activation or differentiation, requiring careful titration of antibody concentrations. Additionally, fixation methods significantly impact detection sensitivity - paraformaldehyde fixation preserves DPP9 epitopes better than methanol for immunofluorescence applications in most cell types. Researchers should validate antibody performance in their specific cell or tissue type of interest and establish appropriate positive and negative controls to accurately interpret staining patterns and expression levels .

How can DPP9 antibodies be used to investigate its role in inflammatory diseases?

DPP9 antibodies provide valuable tools for investigating the protein's role in inflammatory diseases through several methodological approaches. Researchers can perform immunohistochemistry on tissue samples from patients with inflammatory conditions to analyze DPP9 expression levels compared to healthy controls. Flow cytometry with DPP9 antibodies can quantify expression in specific immune cell populations implicated in disease pathogenesis. For mechanistic studies, co-immunoprecipitation using DPP9 antibodies followed by mass spectrometry can identify disease-specific interaction partners. Additionally, proximity ligation assays in patient-derived cells can visualize altered interactions between DPP9 and inflammasome components like NLRP1 and CARD8. Time-course immunoblotting after inflammatory stimuli can track changes in DPP9 expression, phosphorylation, or degradation, revealing dysregulated pathways. These approaches collectively enable researchers to understand how alterations in DPP9 function contribute to inflammasome hyperactivation or dysregulation in conditions like autoinflammatory syndromes, autoimmune disorders, and inflammatory responses to infection .

What technical considerations are important when using DPP9 antibodies in multiplex imaging studies?

Multiplex imaging with DPP9 antibodies requires careful technical optimization to ensure specificity and compatibility with other detection systems. First, antibody selection is critical - use directly conjugated antibodies or primary antibodies from different host species to avoid cross-reactivity in multiplexed detection. Second, perform sequential staining with complete stripping or quenching between rounds if using antibodies from the same species. Third, carefully titrate antibody concentrations to minimize background while maintaining specific signal, as optimal concentrations may differ from single-staining protocols. Fourth, validate spectral unmixing parameters, especially when fluorophores have overlapping emission spectra. Fifth, include appropriate controls, including single-stained samples for each antibody to establish bleed-through profiles and concentration-matched isotype controls. Sixth, consider the order of antibody application, as some epitopes may be more sensitive to harsh elution conditions used in cyclic immunofluorescence. Finally, optimize fixation conditions specifically for multiplex approaches, as some fixatives may preserve DPP9 epitopes better while remaining compatible with other target proteins in your panel .

How should researchers approach validating novel DPP9 antibodies for research applications?

Validating novel DPP9 antibodies requires a comprehensive approach to ensure specificity, sensitivity, and reproducibility across applications. Begin with Western blot validation using positive control samples (human liver tissue lysates) where DPP9 appears at approximately 100-101 kDa, alongside negative controls like DPP9 knockout cells or tissues. Confirm specificity through peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific signals. For application versatility, test performance in multiple techniques including immunofluorescence (using HeLa cells as a positive control with expected cytoplasmic localization), immunoprecipitation, and flow cytometry. Compare the novel antibody's performance with previously validated antibodies targeting different epitopes of DPP9. Assess cross-reactivity with other DPP family members, particularly the closely related DPP8. Evaluate lot-to-lot consistency through side-by-side testing of multiple production lots. Finally, perform functional validation by confirming that the antibody can detect changes in DPP9 expression following siRNA knockdown or overexpression. This systematic validation approach ensures that the antibody will provide reliable results in complex research applications .

What controls are essential when using DPP9 antibodies in various experimental contexts?

When using DPP9 antibodies, a comprehensive set of controls is essential for experimental rigor and data interpretation. For Western blot and immunoprecipitation, include: (1) Positive controls such as human liver tissue lysates where DPP9 appears at approximately 100-101 kDa; (2) Negative controls including DPP9 knockout/knockdown samples; (3) Isotype controls matching the primary antibody's species and isotype; and (4) Loading controls to normalize expression levels. For immunofluorescence and immunohistochemistry, incorporate: (1) Primary antibody omission controls; (2) Secondary antibody-only controls to assess non-specific binding; (3) Known positive cell types (e.g., HeLa cells for cytoplasmic staining); and (4) Peptide competition controls where pre-incubation with immunizing peptide should eliminate specific staining. For functional studies, essential controls include: (1) DPP9 enzymatic activity assays in parallel with antibody detection to correlate protein levels with function; (2) Time-course controls when studying dynamic processes; and (3) Wild-type and catalytically inactive DPP9 controls to distinguish between structural and enzymatic roles of the protein .

How can researchers overcome challenges in detecting endogenous versus overexpressed DPP9?

Detecting endogenous versus overexpressed DPP9 presents distinct challenges requiring specific methodological approaches. For endogenous DPP9 detection, researchers should: (1) Use highly sensitive detection methods like enhanced chemiluminescence or fluorescent secondary antibodies for Western blot; (2) Implement signal amplification techniques such as tyramide signal amplification for immunohistochemistry; (3) Optimize antibody concentration through careful titration experiments; and (4) Employ cell/tissue types known to express higher levels of DPP9 (like liver tissue) as positive controls. For distinguishing endogenous from overexpressed DPP9, researchers can: (1) Use epitope-tagged DPP9 constructs and tag-specific antibodies in parallel with DPP9 antibodies; (2) Perform careful quantitative analysis using standard curves with recombinant DPP9 protein; (3) Design experiments with partial knockdown followed by reconstitution to maintain physiologically relevant expression levels; and (4) Use inducible expression systems to compare the same cells with and without DPP9 overexpression. Additionally, researchers should be aware that overexpression might alter DPP9's localization or interaction partners, potentially affecting antibody accessibility to certain epitopes .

What approaches can help distinguish between DPP9 and other closely related family members?

Distinguishing between DPP9 and other closely related family members, particularly DPP8, requires carefully designed experimental approaches. First, researchers should select antibodies validated for specificity against DPP9's unique epitopes, particularly those targeting regions with minimal sequence homology to other DPP family members. Second, Western blot analysis can help distinguish family members based on molecular weight differences—DPP9 appears at approximately 100-101 kDa, while other family members migrate differently. Third, include recombinant proteins of each family member as controls in immunoassays to confirm antibody specificity. Fourth, implement siRNA or CRISPR/Cas9-mediated knockdown of DPP9 and verify that only DPP9-specific signals are reduced. Fifth, employ immunoprecipitation followed by mass spectrometry to confirm antibody specificity based on peptide signatures unique to DPP9. Sixth, utilize selective inhibitors (where available) to distinguish between family members' enzymatic activities in parallel with antibody-based detection. Finally, compare expression patterns across tissues and cell types, as DPP family members often show distinct tissue distribution profiles, providing contextual evidence for antibody specificity .

How can researchers effectively use DPP9 antibodies to study inflammasome regulation mechanisms?

Researchers can effectively use DPP9 antibodies to study inflammasome regulation through several sophisticated approaches. One powerful method involves proximity-dependent labeling techniques, where DPP9 antibodies validate the spatial relationships between DPP9 and inflammasome components identified through BioID or APEX2 approaches. Another approach utilizes super-resolution microscopy with DPP9 antibodies to visualize nanoscale associations with NLRP1 and CARD8 before and after inflammasome activation. Sequential immunoprecipitation (IP) experiments, first pulling down with DPP9 antibodies followed by NLRP1 or CARD8 antibodies, can isolate the ternary complex for detailed compositional analysis. For functional studies, researchers can combine DPP9 immunodepletion from cell lysates with in vitro inflammasome reconstitution assays to determine how DPP9 removal affects inflammasome assembly kinetics. Additionally, phospho-specific DPP9 antibodies (if available) can track post-translational modifications that might regulate DPP9's inflammasome-inhibitory function. Finally, chromatin immunoprecipitation (ChIP) studies using antibodies against transcription factors that regulate DPP9 expression can connect broader inflammatory signaling networks to DPP9-mediated inflammasome control .

What methodological approaches can help understand temporal changes in DPP9 expression and localization during cellular stress?

To understand temporal changes in DPP9 expression and localization during cellular stress, researchers should implement complementary methodological approaches. Live-cell imaging combined with fluorescently tagged DPP9 antibody fragments (Fabs) or tagged DPP9 can track real-time localization changes, while providing baseline data for comparison with fixed-cell analysis using conventional antibodies. Pulse-chase experiments with metabolic labeling followed by DPP9 immunoprecipitation can determine protein synthesis and degradation rates during stress. For subcellular fractionation studies, DPP9 antibodies can track redistribution between compartments, particularly movement between cytosolic and membrane-associated fractions during stress responses. Quantitative immunofluorescence with automated image analysis allows measurement of DPP9 intensity and colocalization with organelle markers across large cell populations at multiple timepoints. Additionally, proximity ligation assays using DPP9 antibodies paired with stress granule markers or inflammasome components can reveal dynamic protein-protein interactions specific to stress conditions. Finally, phospho-proteomic analysis following DPP9 immunoprecipitation can identify stress-induced post-translational modifications that might regulate its function, localization, or stability .

How can researchers employ DPP9 antibodies to investigate its role in non-canonical functions beyond inflammasome regulation?

Investigating DPP9's non-canonical functions beyond inflammasome regulation requires innovative applications of DPP9 antibodies. Researchers can conduct comparative interactome studies by performing DPP9 immunoprecipitation from different cellular compartments followed by mass spectrometry, revealing compartment-specific interaction partners that might indicate novel functions. ChIP-seq experiments using DPP9 antibodies can identify potential chromatin associations, as several proteases have been found to have nuclear functions. For extracellular roles, researchers can use DPP9 antibodies in enzyme-linked immunosorbent assays (ELISAs) to detect secreted DPP9 in cell culture media or biological fluids, as some intracellular proteases have functional extracellular counterparts. Tissue cross-linking followed by immunoprecipitation can preserve transient or weak interactions that might be missed in conventional co-IP approaches. Additionally, antibody-based protein complementation assays can validate specific protein-protein interactions in living cells. When investigating potential non-enzymatic structural roles, researchers can combine DPP9 immunodepletion with functional reconstitution using catalytically inactive mutants. Finally, spatial proteomics approaches using DPP9 antibodies for immunofluorescence combined with multiplexed staining of cellular structures can reveal unexpected localizations suggesting novel functions beyond the established cytoplasmic role in inflammasome regulation .