PARP14 Antibody

What is PARP14 and why is it significant in immunological research?

PARP14 (poly (ADP-ribose) polymerase family, member 14) is the largest member of the human PARP family with a calculated molecular weight of 203 kDa (observed at approximately 200 kDa in experimental settings). It functions as a critical regulator of multiple pathways involved in immunity, inflammation, and genome stability . PARP14's significance stems from its documented roles in DNA repair mechanisms, B cell regulation, and focal adhesion processes . Research has demonstrated PARP14's importance in facilitating lymphomagenesis driven by persistent c-Myc overexpression, and its vital function in the Myc-imposed developmental block at the pre-B stage of B lymphoid development. Additionally, PARP14 helps transduce IL-4 signals that promote metabolic fitness and survival of mature B cells . These multifaceted functions make PARP14 a critical target for immunological and cancer research.

How do I select the appropriate PARP14 antibody for my research application?

When selecting a PARP14 antibody, consider these critical factors:

• Experimental application compatibility (WB, IHC, ELISA)

• Species reactivity required (human, mouse, or both)

• Antibody class (monoclonal vs. polyclonal; recombinant vs. conventional)

• Validated performance in your specific application

Always confirm antibody performance in your specific experimental system through titration, as optimal performance is sample-dependent . For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative .

How can I effectively use PARP14 antibodies for immunohistochemistry studies?

For optimal IHC results with PARP14 antibodies, follow these methodological considerations:

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary method. If results are suboptimal, try citrate buffer at pH 6.0 as an alternative .

• Antibody dilution: Begin with a dilution range of 1:50-1:500, conducting titration experiments to determine optimal concentrations for your specific tissue sample .

• Positive controls: For human studies, placenta tissue has been validated as a positive control. For mouse studies, colon tissue shows reliable PARP14 expression .

• Blocking and incubation: Standard IHC protocols apply, but ensure thorough blocking of endogenous peroxidase activity and non-specific binding sites.

• Detection system: Choose a detection system compatible with rabbit IgG primary antibodies, as the available PARP14 antibodies are rabbit-derived .

Remember that both polyclonal (30127-1-AP) and recombinant (84039-6-RR) antibodies are available, which may provide different specificity and sensitivity profiles depending on your research question .

What are the most reliable methods for Western blot detection of PARP14?

For reliable Western blot detection of PARP14, consider these methodological approaches:

• Sample preparation: Due to PARP14's large size (203 kDa calculated, 200 kDa observed), use a low percentage gel (6-8%) or gradient gel to achieve proper separation. Ensure complete protein denaturation and use fresh samples to prevent degradation .

• Antibody selection and dilution: The 84039-6-RR antibody has been validated for WB applications at 1:500-1:2000 dilution. Start with a mid-range dilution (1:1000) and adjust as needed .

• Detection system: Use a sensitive detection system capable of visualizing high molecular weight proteins, such as enhanced chemiluminescence.

• Positive controls: A549 and U2OS cell lysates have been confirmed as positive controls for PARP14 expression .

• Expected banding pattern: Look for a primary band at approximately 200 kDa corresponding to full-length PARP14. Be aware of potential degradation products in improperly handled samples .

For optimal results, titrate the antibody in your specific testing system and include appropriate positive controls to validate your findings .

How does PARP14 influence antibody responses, and how can this be studied experimentally?

PARP14 has been demonstrated to influence the class distribution, affinity repertoire, and recall capacity of antibody responses in mice . To study these effects experimentally:

• Experimental models: Compare wild-type and PARP14-deficient (Parp14 −/−) mice in immunization studies using hapten-conjugate systems (e.g., NP-KLH) or pathogen challenge models (e.g., human metapneumovirus) .

• Antibody class analysis: Measure multiple antibody isotypes (IgA, IgE, IgG1, IgG2a, IgG2b) following primary and recall immunizations. Research shows PARP14 deficiency particularly affects IgA and IgE responses while having minimal impact on IgG1 .

• Affinity maturation assessment: Utilize differential binding to hapten densities (e.g., NP2 vs. NP14) to evaluate antibody affinity maturation. PARP14-deficient mice show defects in generating high-affinity antibodies .

• Cellular mechanisms: Implement both in vivo and in vitro approaches to determine whether defects are B-cell intrinsic or extrinsic. For IgE responses, PARP14 functions cell-intrinsically in B cells, while IgA defects involve both B-cell intrinsic mechanisms and helper T cell deficiencies .

• Recall response quantification: Measure the fold increase between primary and recall responses as PARP14's impact is amplified during memory responses (particularly for IgA and IgE) .

This multi-faceted approach allows for comprehensive evaluation of how PARP14 influences different aspects of humoral immunity and memory formation.

What role does PARP14 play in allergic inflammation, and how can researchers investigate this experimentally?

PARP14 plays significant roles in allergic inflammation, particularly affecting IgE production and inflammatory responses. To investigate these roles experimentally:

• Allergic lung inflammation models: Use ovalbumin or other allergen sensitization and challenge models in wild-type versus PARP14-deficient mice. Research shows that PARP14 deficiency results in reduced IgE anti-ovalbumin levels after both primary and recall exposures .

• Cytokine profiling: Measure Th2 and Th17 cytokines in bronchoalveolar lavage fluid and lung tissue. PARP14 deficiency results in reduced cytokine levels, particularly during recall responses .

• Cellular analysis: Quantify airway eosinophilia and other inflammatory cell infiltration. PARP14-deficient mice show reduced eosinophil recruitment during recall challenges .

• In vitro IgE production: Culture purified B cells with IL-4 and anti-CD40 to induce class-switching to IgE. Flow cytometry and ELISA can then be used to measure IgE+ cell frequency and secreted IgE, respectively. PARP14-deficient B cells show reduced capacity for IgE production compared to wild-type cells .

• Mechanistic dissection: Compare results with other gene-deficient models (e.g., Stat6−/−) to position PARP14 within known signaling pathways. This helps determine whether PARP14 functions upstream, downstream, or parallel to established mediators .

These approaches collectively allow researchers to dissect the specific contributions of PARP14 to allergic inflammation at both cellular and molecular levels.

What are common technical challenges when working with PARP14 antibodies and how can they be overcome?

Working with PARP14 antibodies presents several technical challenges:

• High molecular weight detection: At approximately 200 kDa, PARP14 can be difficult to transfer efficiently in Western blots.

  • Solution: Use low percentage gels (6-8%), extend transfer times, and add SDS to transfer buffer to enhance high molecular weight protein transfer .

• Specificity concerns: Distinguishing specific from non-specific signals.

  • Solution: Include positive controls (A549 cells, U2OS cells for Western blot; human placenta or mouse colon tissue for IHC) and negative controls (PARP14 knockout samples where available) .

• Variable expression levels: PARP14 expression may vary across tissues and cell types.

  • Solution: Optimize protein loading amounts, extend exposure times for Western blots, and adjust antibody concentrations based on expected expression levels .

• Antigen accessibility in IHC: Inadequate epitope exposure.

  • Solution: Compare both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0) to determine optimal conditions for your tissue samples .

• Antibody storage stability: Potential loss of activity during storage.

  • Solution: Store antibodies at -20°C in aliquots to avoid repeated freeze-thaw cycles. Products are stable for one year after shipment when properly stored .

For all applications, careful titration of the antibody in your specific experimental system is essential for optimal results .

How should researchers validate PARP14 antibody specificity for their experimental systems?

Rigorous validation of PARP14 antibody specificity is critical for generating reliable research data. Implement these validation approaches:

• Genetic controls: The gold standard for antibody validation is testing in PARP14 knockout or knockdown systems. Compare signal between wild-type and PARP14-deficient samples to confirm specificity .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (approximately 200 kDa for full-length PARP14) .

• Multiple antibody approach: Use antibodies from different sources or those targeting different epitopes of PARP14. Concordant results from multiple antibodies increase confidence in specificity .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide/protein (where available) to demonstrate signal blockade.

• Reproducibility across applications: If detecting PARP14 using multiple techniques (WB, IHC, IF), consistent patterns of expression across methodologies support antibody specificity.

• Positive control samples: Include established PARP14-expressing cell lines (A549, U2OS) or tissues (human placenta, mouse colon) in your validation studies .

• Immunoprecipitation-mass spectrometry: For ultimate validation, immunoprecipitate with the PARP14 antibody and confirm target identity by mass spectrometry.

How is PARP14 involved in immune-related disease models, and what experimental approaches best study these connections?

PARP14 has demonstrated important roles in several immune-related disease contexts through its influence on antibody responses and inflammatory processes:

• Respiratory infection models: Human metapneumovirus (hMPV) infection models reveal that PARP14-deficient mice generate lower levels of virus-specific serum IgA during both primary infection and recall responses. This is evidenced by reduced numbers of anti-MPV IgA-secreting cells in infected lungs of PARP14-null animals .

• Allergic lung inflammation: PARP14 deficiency results in reduced IgE anti-ovalbumin levels, decreased Th2 and Th17 cytokines, and diminished airway eosinophilia, particularly during recall challenges. This indicates PARP14's importance in allergic responses .

• B cell development and antibody production: PARP14 influences class distribution of antibodies, with particular effects on IgA and IgE production. The mechanisms differ by antibody class—IgE defects are B-cell intrinsic, while IgA production involves both B-cell and helper T cell mechanisms .

Experimental approaches to study these connections include:

• Comparative studies of wild-type versus PARP14-deficient mice in infection and inflammation models

• Analysis of primary versus recall immune responses to assess memory development

• Cell-type specific deletion models to distinguish intrinsic versus extrinsic mechanisms

• In vitro class-switching assays to examine direct effects on B cell function

• Adoptive transfer experiments to delineate cell-specific contributions

These approaches collectively enable mechanistic understanding of how PARP14 contributes to immune-related disease processes.

What is currently understood about PARP14's role in cancer biology, and how can researchers investigate this experimentally?

PARP14's roles in cancer biology are emerging as significant research areas:

• Lymphomagenesis: PARP14 facilitates lymphomagenesis driven by persistent overexpression of the oncogene c-Myc. It is vital for the Myc-imposed block at the pre-B stage of B lymphoid development .

• Survival signaling: PARP14 helps transduce IL-4 signals that promote metabolic fitness and survival of B cells, which may contribute to certain B cell malignancies .

• Plasmacytoma survival: Research indicates that PARP14 impacts the survival of plasmacytoma cells, suggesting potential roles in plasma cell malignancies .

• Germinal center regulation: Since c-Myc is a major regulator of germinal center B cells, and PARP14 interacts with Myc signaling, PARP14 may influence germinal center-derived lymphomas .

Experimental approaches to investigate these connections include:

• Lymphoma models in PARP14-deficient versus wild-type backgrounds, particularly those driven by c-Myc overexpression

• Analysis of PARP14 expression and activity in primary human lymphoma samples

• Cell line studies examining PARP14 knockdown/knockout effects on survival, proliferation, and metabolism

• Investigation of potential synthetic lethal interactions with other cancer-related pathways

• Exploration of small molecule inhibitors of PARP14's ADP-ribosyltransferase activity as potential therapeutic approaches

These approaches allow researchers to determine the mechanistic involvement of PARP14 in cancer biology and evaluate its potential as a therapeutic target.

What are emerging research questions regarding PARP14's enzymatic activity and its influence on cellular functions?

Several compelling research questions are emerging regarding PARP14's enzymatic function:

• Substrate specificity: What are the specific protein targets of PARP14's ADP-ribosyltransferase activity in different cellular contexts? Studies utilizing ADP-ribosyltransferase dead mutants of PARP14 (through point mutations in the C-terminal catalytic domain) can help identify enzymatic versus non-enzymatic functions .

• Catalytic mechanism regulation: How is PARP14's enzymatic activity regulated in different immune cell types and under various stimulation conditions? Is its activity constitutive or induced by specific signals?

• NAD+ sensing role: Given that PARP enzymes utilize NAD+ as a substrate, does PARP14 function as a cellular NAD+ sensor, linking metabolic state to immune function?

• Cross-talk with other ADP-ribosylation events: How does PARP14 activity interact with other PARP family members and microbial ADP-ribosylating toxins? Research hints at potential parallels between functions of microbial ADP-ribosylating toxins and intracellular mammalian ADP-ribosyltransferases like PARP14 .

• Structural determinants of function: What structural features of PARP14 determine its unique functions compared to other PARP family members?

Methodological approaches to address these questions include:

• Site-directed mutagenesis of catalytic residues

• Comparative activity assays using radioactive 32P-NAD+ incorporation

• Identification of ADP-ribosylated proteins by mass spectrometry

• Structural studies of PARP14's catalytic domain in complex with substrates

• Pharmacological inhibition with selective small molecules

These approaches will yield critical insights into how PARP14's enzymatic activity contributes to its diverse cellular functions.

How might PARP14-targeting therapeutic approaches be developed and evaluated?

The development of PARP14-targeting therapeutic approaches represents an emerging frontier with several promising research directions:

• Small molecule inhibitor development: Design and screening of small molecules that selectively inhibit PARP14's catalytic activity. This would build upon established methods used for other PARP family members (like PARP1/2 inhibitors used in cancer therapy).

• B cell-specific targeting strategies: Given PARP14's role in B cell functions and antibody production, B cell-directed delivery systems could enhance therapeutic specificity for conditions like allergic disorders or B cell malignancies .

• Combination therapy evaluation: Research suggests PARP14 deficiency impacts efficacy of allergic responses and antibody production. Testing PARP14 inhibitors in combination with existing allergic disease treatments could reveal synergistic effects .

• Disease-specific biomarkers: Identifying biomarkers that predict responsiveness to PARP14-targeting therapies would enable precision medicine approaches.

• Therapeutic window assessment: Comprehensive evaluation of potential side effects based on PARP14's multiple physiological roles, particularly in DNA repair and immune function.

Experimental approaches for evaluating these therapeutics include:

• In vitro enzymatic assays for inhibitor screening

• Cellular models assessing target engagement and functional outcomes

• Animal models of allergic disease, infection, and B cell malignancies

• Safety and toxicity evaluations in relevant pre-clinical models

• Biomarker discovery studies correlating PARP14 expression/activity with disease outcomes

These research directions provide a framework for development of novel therapeutics targeting PARP14 in immune disorders and cancer.