PARP9 Antibody

How do I select the appropriate PARP9 antibody for my research application?

When selecting a PARP9 antibody, consider multiple factors based on your experimental needs:

• Target species reactivity: Different antibodies show reactivity with specific species (human, mouse, rat). For example, product 17535-1-AP shows reactivity with human and rat samples .

• Application compatibility: Verify that the antibody has been validated for your intended application (WB, IHC, IF, IP). For instance, anti-PARP9 antibody ab53796 is suitable for IP and WB applications and reacts with human samples .

• Clonality: Decide between polyclonal (broader epitope recognition) and monoclonal (higher specificity) based on your research needs.

• Validation data: Review existing publications and validation data. Some antibodies like 17535-1-AP have been cited in multiple publications for applications such as KD/KO, WB, and IF .

• Immunogen information: Check if the antibody was raised against a region relevant to your research question. For example, HPA066708 was generated against the immunogen sequence "IDNEVLMAAFQRKKKMMEEKLHRQPVSHRLFQQVPYQFCNVVCRVGFQRMYSTPCDPKYGAGIYFTKNLKNLAEKAKKISAA" .

What validation steps should I perform before using a PARP9 antibody in my experiments?

A comprehensive validation strategy includes:

• Positive and negative controls: Use cell lines with known PARP9 expression (e.g., Raji cells, THP-1 cells demonstrate positive WB signals) .

• Knockdown/knockout verification: Implement PARP9 knockdown/knockout approaches to confirm specificity, as demonstrated in publications using anti-PARP9 antibodies .

• Cross-reactivity assessment: Test for cross-reactivity with other PARP family members, especially similar molecules like PARP14, which has functional interactions with PARP9 .

• Epitope mapping: Understand the specific region of PARP9 recognized by your antibody to ensure it detects the form relevant to your research.

• Multiple technique confirmation: Validate expression using orthogonal methods (e.g., if using for IHC, confirm with WB).

• Titration optimization: Determine optimal working dilutions for your specific application (e.g., HPA066708 recommended dilutions: WB 0.04-0.4 μg/mL, IF 0.25-2 μg/mL) .

How can I optimize Western blot protocols for PARP9 detection?

Optimizing Western blot for PARP9 detection requires specific considerations:

• Expected molecular weight: PARP9 has an observed molecular weight of approximately 88-92 kDa , so ensure your gel resolution is appropriate for this range.

• Sample preparation:

  • For cell lysates, use RIPA buffer with protease inhibitors and phosphatase inhibitors (particularly important if studying PARP9 phosphorylation or interaction with PI3K/AKT pathway) .

  • Include sonication steps to ensure complete nuclear protein extraction, as PARP9 functions in DNA repair .

• Antibody dilution: Start with manufacturer's recommended dilution (e.g., 1:2000-1:12000 for 17535-1-AP) and adjust as needed.

• Blocking conditions: 5% non-fat milk in TBST for 1 hour at room temperature generally works well, but BSA may be preferred when studying phosphorylation.

• Signal detection: For detecting potentially low levels of endogenous PARP9, consider using enhanced chemiluminescence or fluorescent secondary antibodies to improve sensitivity.

• Positive controls: Include lysates from Raji cells or THP-1 cells, which have been demonstrated to express detectable levels of PARP9 .

• Stripping and reprobing: If examining pathway components, consider gentle stripping methods to probe for interacting partners like DTX3L or signaling molecules in the PI3K/AKT pathway .

What are the best approaches for immunohistochemical detection of PARP9 in tissue samples?

For optimal IHC detection of PARP9:

• Tissue processing and fixation: Use 10% neutral-buffered formalin fixation for 24-48 hours, as overfixation may mask epitopes.

• Antigen retrieval:

  • Use TE buffer pH 9.0 for optimal results with many PARP9 antibodies .

  • Alternative citrate buffer pH 6.0 may be tested if TE buffer yields suboptimal results.

• Antibody dilution: Start with recommended dilution (e.g., 1:20-1:200 for 17535-1-AP) and optimize for your specific tissue type.

• Positive control tissues: Include spleen, skin, kidney, heart, lung, or ovary tissues as positive controls, as these have demonstrated PARP9 expression .

• Counterstaining: Hematoxylin counterstaining provides contrast for nuclear visualization, important as PARP9 may show both nuclear and cytoplasmic localization.

• Co-staining considerations: For macrophage-specific PARP9 detection in granulomas or tumor tissues, consider dual staining with CD68 as PARP9 has been shown to co-localize with CD68+ macrophages in tuberculosis granulomas .

• Image analysis: Quantify PARP9 expression using digital pathology approaches, particularly for comparative studies between different tissue conditions or disease states.

How should I interpret PARP9 expression patterns in different disease contexts?

Interpretation of PARP9 expression requires context-specific considerations:

• Cancer contexts:

  • In gliomas, high PARP9 expression correlates with poor prognosis and advanced clinicopathological features . Compare expression levels with tumor grade and clinical outcomes.

  • In breast cancer, evaluate PARP9 in relation to chemoresistance mechanisms, particularly through the PI3K/AKT/PD-L1 axis .

  • Analyze subcellular localization differences between tumor and normal tissues.

• Immune response contexts:

  • In infectious diseases like tuberculosis, PARP9 expression in CD68+ macrophages differs between controllers and progressors . Quantify expression in relation to disease progression markers.

  • For viral infections, evaluate PARP9 in relation to type I interferon responses and PI3K/AKT pathway activation .

• Comparative analysis recommendations:

  • Use standardized scoring systems (H-score, percentage positive cells, intensity scales).

  • Implement quantitative image analysis with appropriate controls and statistical methods.

  • Consider single-cell approaches to resolve heterogeneity in expression patterns.

How do I distinguish between PARP9 and other PARP family members in my data analysis?

Distinguishing between PARP family members requires attention to:

• Molecular weight differentiation: PARP9 has an observed molecular weight of ~88-92 kDa , which differs from other family members (e.g., PARP1: 116 kDa, PARP14: 170-200 kDa).

• Expression pattern analysis:

  • PARP9 often shows coordinated expression with its binding partner DTX3L , which can serve as a confirmatory marker.

  • PARP9 and PARP14 show functional interactions but have distinct expression patterns in inflammatory conditions .

• Functional validation approaches:

  • Use specific PARP9 knockdown/knockout to confirm phenotypes attributed to PARP9 activity.

  • Implement domain-specific mutants to distinguish between PARP9 macrodomain functions versus catalytic activities.

• Bioinformatic analysis strategies:

  • Perform correlation analysis between PARP9 and inflammatory gene signatures, as PARP9 shows strong correlation with HCK, LCK, interferon, STAT1, MHC I, and MHC II clusters, but negative correlation with IgG clusters .

  • Use gene set enrichment analysis to identify PARP9-associated pathways, which typically include antigen processing, B-cell receptor signaling, cytokine-receptor interactions, and JAK-STAT signaling .

How does PARP9 function in the context of DNA damage repair, and what methods can detect this activity?

PARP9's role in DNA damage repair involves several mechanisms that can be investigated using the following approaches:

• PARP9-DTX3L complex analysis:

  • Co-immunoprecipitation to detect PARP9 interaction with DTX3L and recruitment to DNA damage sites .

  • Proximity ligation assay to visualize PARP9-DTX3L interactions in situ following DNA damage.

• Recruitment dynamics assessment:

  • Live-cell imaging with fluorescently tagged PARP9 to track recruitment kinetics to laser-induced DNA damage sites.

  • ChIP-seq to map PARP9 binding at DNA damage sites genome-wide.

• Functional readouts:

  • Measure recruitment of DNA repair proteins (53BP1, BRCA1, RAP80) to damage sites in PARP9-depleted versus control cells .

  • Assess non-homologous end joining (NHEJ) efficiency using reporter assays in PARP9-modified cells .

• Mechanistic considerations:

  • Investigate PARP9 mono-ADP-ribosylation activity on ubiquitin, which affects ubiquitin conjugation to histones during DNA repair .

  • Examine PARP9's interaction with ribosylated PARP1 at damage sites using specific antibodies against ADP-ribosylated PARP1 .

What are the most effective experimental approaches to study PARP9's role in immune response regulation?

To investigate PARP9's immunoregulatory functions:

• Macrophage-specific studies:

  • Use bone marrow-derived macrophages from wild-type and Parp9-/- mice to compare cytokine responses to pathogen stimulation (demonstrated differences in IFN-β, IL-10, IL-1α, IL-1β production) .

  • Implement CRISPR-Cas9 knockout of PARP9 in human macrophage cell lines to assess impact on inflammatory responses.

• Type I IFN pathway analysis:

  • Use reporter assays (ISRE-luciferase) to measure type I IFN activity in PARP9-modified cells.

  • Perform phospho-flow cytometry to evaluate STAT1/STAT6 phosphorylation states, as PARP9 regulates STAT1 ADP-ribosylation .

• RNA sensing mechanisms:

  • RNA immunoprecipitation (RIP) to detect direct binding between PARP9 and viral RNA .

  • In vitro binding assays with purified PARP9 and synthetic RNA molecules to characterize binding specificity.

• PI3K/AKT pathway activation:

  • Western blot analysis of phosphorylated PI3K and AKT in response to RNA stimulation in PARP9-depleted versus control cells .

  • Use PI3K inhibitors to determine if PARP9-dependent responses require PI3K/AKT signaling.

• In vivo infection models:

  • Compare susceptibility to viral or bacterial infection in wild-type versus Parp9-/- mice .

  • Analyze tissue-specific immune cell infiltration and cytokine profiles during infection.

What are the common technical challenges when working with PARP9 antibodies, and how can they be overcome?

Researchers frequently encounter these challenges when working with PARP9 antibodies:

• Non-specific binding:

  • Problem: Detection of bands at unexpected molecular weights.

  • Solution: Use PARP9 knockout/knockdown controls to identify specific bands; optimize antibody concentration; increase washing steps; consider alternative blocking agents (BSA vs. milk).

• Low signal intensity:

  • Problem: Weak detection of endogenous PARP9.

  • Solution: Implement signal amplification methods; concentrate protein samples; extend primary antibody incubation time (overnight at 4°C); use enhanced detection reagents.

• Nuclear protein extraction difficulties:

  • Problem: Incomplete extraction of nuclear PARP9.

  • Solution: Include nuclear lysis steps with sonication; use specialized nuclear extraction buffers with higher salt concentrations; ensure complete cell lysis verification.

• Inconsistent immunohistochemistry results:

  • Problem: Variable staining across tissue samples.

  • Solution: Standardize fixation times; optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0) ; use automated staining platforms; implement tissue microarrays for consistency.

• Cross-reactivity with other PARP family members:

  • Problem: Difficulty distinguishing PARP9 from related proteins.

  • Solution: Verify specificity using PARP9-knockout samples; perform peptide competition assays; consider using multiple antibodies targeting different epitopes.

How can I address contradictory results in PARP9 function between different experimental models?

When facing contradictory PARP9 functional data:

• Context-dependent role reconciliation:

  • In viral infections, PARP9 acts as a positive regulator of type I IFN responses , while in tuberculosis, it functions as a negative regulator . This apparent contradiction reflects context-specific functions.

  • Solution: Carefully document experimental conditions (cell types, stimulation protocols, timing) and incorporate pathway-specific readouts.

• Model system variations:

  • PARP9 may show different effects in human versus mouse systems or in different cell types.

  • Solution: Validate findings across multiple model systems; use primary cells when possible; compare results from cell lines to primary tissue samples.

• Temporal dynamics considerations:

  • PARP9 functions may differ at early versus late timepoints in immune responses.

  • Solution: Implement time-course experiments; use inducible expression/deletion systems to control timing of PARP9 modulation.

• Interaction partner dependencies:

  • PARP9 functions often depend on its binding partner DTX3L .

  • Solution: Assess DTX3L expression/function concurrently; use co-expression or co-depletion approaches to study the complex rather than PARP9 alone.

• Pathway cross-talk analysis:

  • PARP9 interacts with multiple pathways (DNA repair, PI3K/AKT, STAT signaling) .

  • Solution: Use systems biology approaches to model pathway interactions; implement combinatorial inhibition of multiple pathways.

How can single-cell approaches be applied to study PARP9 function in heterogeneous tissues?

Single-cell methodologies offer unique insights into PARP9 biology:

• Single-cell RNA sequencing (scRNA-seq):

  • Can reveal cell type-specific expression patterns of PARP9 and co-regulated genes in complex tissues like tumors or inflammatory lesions.

  • Implementation strategy: Compare PARP9 expression across immune cell subsets in glioma microenvironment, as PARP9 shows correlation with immune infiltration .

• Single-cell proteomics:

  • Enables quantification of PARP9 protein levels and post-translational modifications at single-cell resolution.

  • Method: Mass cytometry (CyTOF) with validated PARP9 antibodies allows simultaneous detection of PARP9 and signaling pathway components.

• Spatial transcriptomics:

  • Preserves tissue context while providing transcriptional information about PARP9 and related genes.

  • Application: Map PARP9 expression in relation to tissue structures like tumor margins or granulomas, where PARP9+ macrophages show differential distribution .

• Multiparameter imaging:

  • Enables visualization of PARP9 alongside multiple markers to understand cellular interactions.

  • Approach: Multiplex immunofluorescence or imaging mass cytometry to analyze PARP9 co-expression with CD68 and activation markers in tuberculosis granulomas .

• Analytical considerations:

  • Implement trajectory analysis to identify transitions in PARP9 expression during cellular differentiation or disease progression.

  • Use computational deconvolution to infer PARP9-associated signatures from bulk tissue data.

What are the key considerations for developing and validating PARP9 inhibitors for research applications?

For researchers developing PARP9-targeted compounds:

• Target specificity assessment:

  • Challenge: PARP9 shares structural similarities with other PARP family members.

  • Approach: Implement in vitro activity assays against a panel of recombinant PARP enzymes; use structural biology (crystallography, cryo-EM) to guide selective inhibitor design.

• Domain-specific targeting strategies:

  • PARP9 contains macrodomains with ADPr hydrolase activity and regulatory functions .

  • Approach: Design domain-specific inhibitors targeting either macrodomain 1 (with hydrolytic activity) or macrodomain readers to dissect different PARP9 functions.

• Complex-based considerations:

  • PARP9 functions in complex with DTX3L .

  • Strategy: Target the PARP9-DTX3L interface to disrupt the complex rather than individual enzymatic activities.

• Cellular validation methods:

  • Verify target engagement using cellular thermal shift assays (CETSA).

  • Evaluate functional consequences using pathway-specific readouts (DNA damage repair efficiency, IFN signaling outputs).

• In vivo evaluation approaches:

  • Pharmacokinetic/pharmacodynamic studies should include biomarkers of PARP9 activity inhibition.

  • Consider disease models where PARP9 has established roles (cancer chemoresistance , inflammatory conditions ) for therapeutic potential assessment.

• Potential therapeutic applications:

  • For cancer therapy: Target PARP9 to overcome chemoresistance in breast cancer .

  • For infectious diseases: Modulate PARP9 to enhance antiviral responses or reduce hyperinflammation in tuberculosis .