DIR9 Antibody

What is DPP9 and where is it typically expressed in human tissues?

DPP9 (Dipeptidyl Peptidase 9) is a serine protease belonging to the S9B protein family. It is widely expressed in human tissues, with notable detection in liver tissue as demonstrated by Western blot analysis using anti-DPP9 monoclonal antibodies. Subcellular localization studies using immunofluorescence techniques have shown that DPP9 is predominantly localized to the cytoplasm, as observed in HeLa cervical epithelial carcinoma cells . DPP9 plays roles in cell signaling, immune function, and protein processing pathways. Recent research has also implicated DPP9 in inflammasome regulation pathways, suggesting its importance in innate immunity responses.

What is the molecular weight of human DPP9 when detected by antibodies?

Western blot analysis using anti-human DPP9 monoclonal antibodies typically detects a specific band at approximately 100 kDa in human liver tissue lysates. When using Simple Western analysis (an automated capillary-based size separation technique), DPP9 is detected at approximately 101 kDa. This slight variation is within the expected technical range for different detection methods . Researchers should be aware that the predicted molecular weight from amino acid sequence (Arg2-Leu892) may differ slightly from observed mobility on electrophoretic gels due to post-translational modifications.

What are the primary applications for DPP9 antibodies in research?

DPP9 antibodies have been validated for several common research applications:

• Western Blot Analysis: Successfully used to detect DPP9 in human liver tissue lysates at concentrations of approximately 1 μg/mL under reducing conditions.

• Immunocytochemistry: Effective for detecting DPP9 in fixed cells, such as HeLa cells, typically at concentrations around 10 μg/mL.

• Simple Western Analysis: Automated capillary-based detection of DPP9 in tissue lysates.

• Immunoprecipitation: Used in studies examining protein-protein interactions, as referenced in research examining the ZAK/P38 kinase signaling pathway .

The optimal antibody concentration should be determined empirically for each application and sample type.

How can I ensure specificity when using DPP9 antibodies in my research?

Ensuring antibody specificity is critical for generating reliable results. For DPP9 antibodies, consider these validation approaches:

• Multiple Detection Methods: Compare results between Western blot, immunofluorescence, and other methods to confirm consistent patterns of detection.

• Positive Controls: Include known DPP9-expressing samples (such as human liver tissue) as positive controls in experiments.

• Negative Controls: Use tissues or cell lines with low or no DPP9 expression, or employ knockdown/knockout approaches (siRNA or CRISPR) to demonstrate specificity.

• Cross-reactivity Testing: Test for potential cross-reactivity with other DPP family members (particularly DPP4 and DPP8) which share structural similarities with DPP9.

• Epitope Mapping: Consider the specific epitope recognized by the antibody (e.g., Arg2-Leu892 region of human DPP9) and assess potential for cross-reactivity with similar sequences .

When using monoclonal antibodies like the 757004 clone, researchers can generally expect greater specificity than with polyclonal antibodies, though thorough validation remains essential.

What is known about DPP9's role in inflammasome regulation pathways?

Recent research has implicated DPP9 in inflammasome regulation, particularly in the context of hematopoiesis. Studies examining the ZAK/P38 kinase signaling pathway have demonstrated that this pathway regulates hematopoiesis by activating the NLRP1 inflammasome, and DPP9 appears to play a role in this process . Researchers investigating DPP9 in this context should consider:

• Pathway Analysis: Examining interactions between DPP9 and other components of the NLRP1 inflammasome complex.

• Functional Studies: Assessing how DPP9 inhibition or depletion affects inflammasome activation and downstream cytokine production.

• Cell Type Specificity: Determining whether DPP9's role in inflammasome regulation varies across different cell types, particularly in hematopoietic versus non-hematopoietic cells.

This represents an emerging area of research where DPP9 antibodies serve as crucial tools for elucidating protein-protein interactions and signaling mechanisms.

How can epitope mapping approaches improve DPP9 antibody experimental design?

Understanding epitope binding characteristics is increasingly important in antibody research. As demonstrated in SARS-CoV-2 antibody studies, epitope mapping using phage display technology can reveal critical insights about antibody binding properties . For DPP9 research, similar approaches could:

• Identify Critical Binding Regions: Determine precisely which amino acid sequences within DPP9 are recognized by specific antibodies.

• Predict Cross-Reactivity: Assess potential cross-reactivity with other proteins sharing similar epitope sequences.

• Optimize Detection Methods: Tailor experimental conditions based on epitope accessibility in different sample preparation methods.

• Improve Reagent Selection: Select antibodies targeting different epitopes for complementary detection approaches.

Using phage-displayed peptide libraries or similar techniques could generate high-resolution maps of DPP9 epitopes, similar to approaches used in characterizing SARS-CoV-2 antibody responses .

What are the optimal conditions for Western blot detection of DPP9?

Based on published protocols, the following conditions are recommended for optimal Western blot detection of DPP9:

• Sample Preparation: Use reducing conditions for lysate preparation from tissues or cells.

• Antibody Concentration: 1 μg/mL of mouse anti-human DPP9 monoclonal antibody has been validated for Western blot detection.

• Membrane Type: PVDF membranes show good results for DPP9 detection.

• Secondary Antibody: HRP-conjugated anti-mouse IgG secondary antibody works effectively with the monoclonal anti-DPP9 antibody.

• Expected Band Size: Look for a specific band at approximately 100 kDa.

• Buffer Systems: Immunoblot Buffer Group 1 has been successfully used in published protocols .

The specific protocol may need optimization depending on sample type and experimental goals.

What considerations are important for immunofluorescence detection of DPP9?

For successful immunofluorescence detection of DPP9 in cell cultures:

• Fixation Method: Immersion fixation has been successfully used for DPP9 detection in HeLa cells.

• Antibody Concentration: 10 μg/mL of mouse anti-human DPP9 monoclonal antibody has proven effective.

• Incubation Conditions: 3 hours at room temperature has been validated for primary antibody incubation.

• Secondary Antibody Selection: Fluorescently-labeled anti-mouse IgG antibodies (such as NorthernLights 557-conjugated anti-mouse IgG) provide good visualization.

• Nuclear Counterstaining: DAPI can be used effectively as a nuclear counterstain.

• Expected Localization: DPP9 typically shows cytoplasmic localization in HeLa cells .

Researchers should also consider including appropriate controls and optimizing the protocol for specific cell types of interest.

How can computational approaches assist in antibody research and design?

Recent advances in computational biology offer powerful tools for antibody research:

• Structure Prediction: Tools like H3-OPT can predict antibody structures with high accuracy, particularly for challenging regions like the CDR-H3 loop. This allows researchers to better understand binding characteristics and design improved antibodies .

• Epitope Mapping: Computational approaches combined with experimental data can map epitopes on target proteins, helping researchers select antibodies that recognize specific regions of interest.

• Binding Affinity Prediction: Machine learning models can predict binding affinities between antibodies and their targets, potentially reducing experimental screening efforts.

• Cross-Reactivity Assessment: Computational tools can help predict potential cross-reactivity based on structural similarities between proteins, improving antibody specificity.

These computational approaches complement experimental methods like phage display and can accelerate antibody development and characterization . For DPP9 research specifically, such tools could help design antibodies targeting functional domains of the protein.

What troubleshooting approaches should I consider when working with DPP9 antibodies?

When encountering challenges with DPP9 antibody experiments, consider these methodological troubleshooting steps:

• Non-specific Binding: If multiple bands appear in Western blot:

  • Increase blocking time/concentration

  • Optimize antibody dilution

  • Include additional washing steps

  • Note that with Simple Western analysis, non-specific interaction with the 230 kDa standard has been observed

• Weak Signal:

  • Increase antibody concentration

  • Extend incubation time

  • Enhance detection system sensitivity

  • Optimize sample preparation to preserve epitope integrity

• Inconsistent Results:

  • Standardize lysate preparation methods

  • Validate antibody performance with positive control samples

  • Maintain consistent experimental conditions

  • Consider lot-to-lot variations in antibody performance

• Background Issues in Immunofluorescence:

  • Optimize blocking conditions

  • Reduce antibody concentration

  • Include additional washing steps

  • Consider autofluorescence controls

Document all optimization steps methodically to establish a reliable protocol for your specific experimental system.