Cleaved-PARP1 (G215) Antibody

What is Cleaved-PARP1 (G215) Antibody and what specific epitope does it recognize?

Cleaved-PARP1 (G215) Antibody specifically detects endogenous levels of PARP1 protein fragments resulting from proteolytic cleavage adjacent to glycine 215 during apoptosis. This rabbit polyclonal antibody is typically generated against synthesized peptides derived from human PARP1 within the amino acid range 196-245 . The antibody recognizes the 89-kDa C-terminal fragment that forms when caspases cleave PARP1 between aspartic acid 214 and glycine 215, making it an excellent marker for detecting apoptotic cells .

Unlike antibodies against total PARP1, the Cleaved-PARP1 (G215) Antibody selectively binds to the neo-epitope exposed only after caspase-mediated proteolysis, providing a clean readout of apoptotic activity without detecting the intact 116-kDa protein. This specificity makes it valuable for distinguishing between healthy and apoptotic cells in various experimental systems .

What is the molecular significance of PARP1 cleavage during apoptosis?

PARP1 cleavage represents a hallmark biochemical feature of apoptosis and serves multiple functional purposes in programmed cell death:

• Enzymatic inactivation: Cleavage by caspases 3 and 7 inactivates PARP1's catalytic activity, preventing DNA repair and conserving cellular ATP that would otherwise be consumed during PARP1's repair functions .

• Generation of functional fragments: The proteolysis produces two specific fragments with distinct roles:

  • The 89-kDa catalytic fragment has greatly reduced DNA binding capacity and translocates from the nucleus to the cytosol .

  • The 24-kDa DNA-binding domain fragment remains in the nucleus, irreversibly binding to DNA strand breaks where it acts as a trans-dominant inhibitor of active PARP1 and other DNA repair enzymes .

• Prevention of energy depletion: By inhibiting PARP1's ADP-ribosylation activity, cleavage prevents NAD+ and ATP depletion, allowing the energy-dependent apoptotic process to proceed to completion rather than switching to necrotic cell death .

This cleavage event represents a deliberate disabling of cellular repair mechanisms during apoptosis, ensuring that the cell death program proceeds efficiently.

What are the optimal conditions and dilutions for using Cleaved-PARP1 (G215) Antibody in different experimental applications?

For Western blot applications, researchers should use 8-10% polyacrylamide gels to achieve optimal separation of the 89-kDa cleaved fragment . When preparing samples, include protease inhibitors to prevent additional proteolysis after cell lysis. Most protocols recommend loading 20-50 μg of total protein per lane for clear detection of the cleaved fragment .

What positive controls should be used when validating Cleaved-PARP1 (G215) Antibody specificity?

Validating antibody specificity is crucial for reliable experimental results. Recommended positive controls include:

• Jurkat or HeLa cells treated with apoptosis inducers:

  • Staurosporine (1-2 μM for 3-4 hours)

  • Etoposide (25 μM for 3 hours)

  • Actinomycin D

• Experimental design for validation:

  • Include both treated (apoptotic) and untreated samples to visualize the difference between cleaved and uncleaved PARP1.

  • Add a treatment group with caspase inhibitor (e.g., Z-VAD-FMK) to confirm that PARP1 cleavage is caspase-dependent.

  • Consider using PARP1 knockdown cells as a negative control to confirm antibody specificity.

The cleaved PARP1 should appear as a distinct band at approximately 89 kDa in Western blot applications, while this band should be absent or significantly reduced in non-apoptotic or caspase-inhibited samples . Some antibodies may also detect the 24-kDa fragment, though this is less common due to its smaller size and potential for rapid degradation.

How can I optimize sample preparation for maximum detection sensitivity?

Optimal sample preparation is critical for detecting cleaved PARP1 with high sensitivity. Follow these methodological recommendations:

• Cell collection: For adherent cells, collect both floating (potentially apoptotic) and attached cells to avoid bias in your results. Gently scrape cells rather than using harsh enzymatic detachment methods that might affect protein integrity.

• Lysis conditions:

  • Use a lysis buffer containing protease inhibitors (complete cocktail) to prevent further protein degradation.

  • Include phosphatase inhibitors if planning to analyze phosphorylation events alongside PARP1 cleavage.

  • Perform lysis on ice and process samples quickly to minimize protein degradation.

  • RIPA buffer or NP-40 based buffers work well for extracting nuclear proteins like PARP1.

• Sample handling:

  • Freshly prepared samples typically yield better results than frozen-thawed samples.

  • If freezing is necessary, snap-freeze aliquots in liquid nitrogen and store at -80°C.

  • Avoid repeated freeze-thaw cycles of protein samples.

• Protein quantification: Ensure equal loading (20-50 μg per lane) using a reliable protein quantification method like BCA or Bradford assay that is compatible with your lysis buffer components.

• Denaturation conditions: Heat samples at 95°C for 5 minutes in Laemmli buffer containing DTT or β-mercaptoethanol to ensure complete protein denaturation and optimal antibody binding to the cleaved epitope .

How can Cleaved-PARP1 (G215) Antibody be used to distinguish between different cell death mechanisms?

Cleaved-PARP1 (G215) Antibody serves as a powerful tool for differentiating between apoptosis and other cell death pathways, particularly parthanatos (PARP1-dependent cell death):

• Apoptosis vs. Parthanatos discrimination:

  • In apoptosis: PARP1 is cleaved by caspases, generating the specific 89-kDa fragment recognized by Cleaved-PARP1 (G215) Antibody.

  • In parthanatos: PARP1 is hyperactivated but not cleaved in the same caspase-dependent manner .

• Experimental approach:

  • Combine Cleaved-PARP1 (G215) Antibody detection with assays for poly(ADP-ribose) (PAR) accumulation.

  • Include treatments with specific inhibitors:

    • Caspase inhibitors (e.g., Z-VAD-FMK) will prevent PARP1 cleavage in apoptosis

    • PARP inhibitors (e.g., ABT-888) will block PAR formation in parthanatos

  • Monitor subcellular localization of key proteins: the 24-kDa PARP1 fragment remains nuclear in apoptosis, while in parthanatos, full-length PARP1 may show altered distribution patterns .

• Advanced analysis: Combining Cleaved-PARP1 detection with assays for mitochondrial membrane potential, caspase activation, and AIF translocation provides a comprehensive assessment of the specific cell death pathway activated in your experimental system .

Understanding these distinctions is crucial for research into disease mechanisms and therapeutic interventions targeting specific cell death pathways.

What is the relationship between PARP1 cleavage and its role in DNA damage response?

The relationship between PARP1 cleavage and DNA damage response represents a critical regulatory mechanism in cell fate decisions:

• PARP1's normal function in DNA repair:

  • Intact PARP1 functions as a DNA damage sensor that binds to DNA breaks.

  • Upon binding, it catalyzes poly(ADP-ribosyl)ation of various nuclear proteins, including itself, to recruit DNA repair machinery .

  • This process consumes NAD+ to build polymers with average chain lengths of 20-30 ADP-ribose units .

• Impact of cleavage on DNA repair:

  • Caspase-mediated cleavage separates PARP1's DNA-binding domain (24-kDa) from its catalytic domain (89-kDa).

  • The 24-kDa fragment remains bound to DNA breaks but cannot initiate repair, effectively blocking access to these sites by other repair enzymes .

  • The 89-kDa fragment loses its efficient DNA-binding capability and translocates to the cytoplasm, preventing further DNA repair activity .

• Mechanistic significance:

  • This deliberate inactivation of DNA repair during apoptosis prevents futile energy expenditure.

  • It also ensures that the genomic DNA fragmentation characteristic of apoptosis proceeds unimpeded by repair mechanisms.

  • The translocated 89-kDa fragment with attached PAR can mediate additional signaling events in the cytoplasm, including AIF release from mitochondria .

Researchers investigating DNA damage responses should consider using both Cleaved-PARP1 (G215) Antibody and antibodies against total PARP1 to distinguish between repair-competent and repair-deficient cellular states.

How can Cleaved-PARP1 (G215) Antibody be used to study the kinetics of apoptosis in experimental models?

Cleaved-PARP1 (G215) Antibody enables detailed investigation of apoptotic kinetics through several methodological approaches:

• Time-course analysis:

  • Design experiments with samples collected at regular intervals (e.g., 0, 2, 4, 8, 12, 24 hours) after treatment with apoptotic stimuli.

  • Process samples for Western blotting using Cleaved-PARP1 (G215) Antibody to detect the appearance and accumulation of the 89-kDa fragment.

  • Quantify the ratio of cleaved to uncleaved PARP1 at each time point using densitometry.

• Multi-parameter apoptosis detection:

  • Correlate PARP1 cleavage with other apoptotic markers in parallel samples:

    • Caspase-3/7 activity using fluorogenic substrates

    • Phosphatidylserine externalization (Annexin V binding)

    • DNA fragmentation (TUNEL assay)

  • This approach establishes the temporal relationship between different apoptotic events.

• Dose-response relationships:

  • Treat cells with increasing concentrations of apoptotic stimuli and assess PARP1 cleavage at a fixed time point.

  • Alternatively, use a fixed concentration and measure cleavage at different time points to determine both concentration and time dependencies.

• Advanced applications:

  • Combine with flow cytometry to correlate PARP1 cleavage with cell cycle phase.

  • Use in drug response studies to determine the timing and extent of apoptosis induction by experimental therapeutics.

  • Apply in tissue sections for spatial analysis of apoptosis in complex tissues or disease models.

This kinetic information is valuable for understanding the progression of apoptosis in various pathological conditions and for evaluating the effects of potential therapeutic interventions .

What is the significance of the newly discovered role of cleaved PARP1 fragments in parthanatos?

Recent research has uncovered an unexpected role for the 89-kDa PARP1 fragment in mediating cross-talk between apoptotic and parthanatic cell death pathways:

• Novel finding: The 89-kDa PARP1 fragment can be poly(ADP-ribosyl)ated before or during caspase cleavage, generating a modified fragment that serves as a cytoplasmic PAR carrier .

• Mechanistic significance:

  • In the cytoplasm, AIF (apoptosis-inducing factor) binds to PAR polymers attached to the 89-kDa PARP1 fragment.

  • This interaction facilitates AIF release from mitochondria and its translocation to the nucleus, where it can induce large-scale DNA fragmentation.

  • The 89-kDa fragment thus acts as a molecular bridge between the caspase-dependent apoptotic pathway and the PAR-dependent parthanatos pathway .

• Experimental evidence:

  • Studies have shown that staurosporine and actinomycin D treatment can induce both PARP1 auto-poly(ADP-ribosyl)ation and fragmentation.

  • The poly(ADP-ribosyl)ated 89-kDa PARP1 fragments translocate to the cytoplasm, while 24-kDa fragments remain associated with DNA lesions .

  • In the cytoplasm, AIF binding to PAR attached to the 89-kDa fragment facilitates its translocation to the nucleus .

• Research implications:

  • This discovery challenges the traditional view that PARP1 cleavage simply inactivates its function in parthanatos.

  • It suggests that therapeutic strategies targeting either pathway may have unexpected effects on the other.

  • Researchers should consider using both anti-PAR antibodies and Cleaved-PARP1 (G215) Antibody in their studies to fully understand the interplay between these death mechanisms.

This finding represents a significant advancement in our understanding of cell death mechanisms and highlights the complex roles that PARP1 and its fragments play in determining cell fate decisions .

What are the key differences between antibodies recognizing cleaved PARP1 at D214/G215 versus those recognizing total PARP1?

Understanding the distinctions between cleaved-specific and total PARP1 antibodies is essential for experimental design and data interpretation:

For maximum information in research applications:

• Use both antibody types in parallel experiments or on duplicate blots.

• Calculate the cleaved/total PARP1 ratio for quantitative assessment of apoptosis progression.

• Consider using sandwich ELISA kits that can detect both cleaved and total PARP1 simultaneously .

Be aware that in some experimental systems, PARP1 may undergo alternative cleavage by proteases other than caspases, generating fragments of different sizes that may not be detected by D214/G215-specific antibodies .

How should I interpret discrepancies between expected and observed molecular weights when using Cleaved-PARP1 (G215) Antibody?

Discrepancies between expected and observed molecular weights are common in Western blot analysis of cleaved PARP1 and require careful interpretation:

• Expected fragment sizes:

  • The main caspase-generated fragments are 89-kDa (C-terminal) and 24-kDa (N-terminal).

  • These sizes are approximate and may vary slightly depending on the gel system and molecular weight standards used.

• Common sources of discrepancy:

  • Post-translational modifications: The presence of poly(ADP-ribosyl)ation on the cleaved fragment can significantly increase its apparent molecular weight on SDS-PAGE .

  • Alternative cleavage sites: PARP1 can be cleaved by other proteases at different sites, generating fragments of different sizes .

  • Partial degradation: Sample handling issues may lead to additional proteolysis and smaller fragments.

  • Gel system variations: Different gel concentrations and buffer systems can affect protein migration.

• Interpretation guidelines:

  • If the observed band is larger than expected (>89 kDa), consider the possibility of post-translational modifications like PAR chains attached to the cleaved fragment .

  • If multiple bands are observed, compare with positive controls and consider the possibility of alternative cleavage events or partial degradation.

  • When in doubt, confirm results with an alternative antibody targeting a different epitope of cleaved or total PARP1.

• Technical note: Some manufacturers specifically note that the observed molecular weight may differ from theoretical calculations. For example, Elabscience reports that their Cleaved-PARP1 (G215) antibody detects an 89-kDa band despite the calculated MW of PARP1 being 113 kDa .

Understanding these potential variations is crucial for accurate data interpretation in apoptosis research.

How can Cleaved-PARP1 (G215) Antibody be applied in studying PARP1 condensates and their role in DNA repair?

Recent research has revealed that PARP1 undergoes phase separation to form biomolecular condensates that play crucial roles in DNA repair processes. Cleaved-PARP1 (G215) Antibody can be applied to investigate these emerging concepts:

• PARP1 condensate formation and function:

  • PARP1 can form spherical, liquid-like condensates triggered by DNA damage .

  • These condensates partition DNA repair proteins and influence repair pathway choice.

  • Auto-poly(ADP-ribosyl)ation enhances PARP1 condensation, with condensate size proportional to PAR chain length .

• Impact of caspase cleavage on condensates:

  • Cleaved-PARP1 (G215) Antibody can be used to investigate how caspase cleavage affects PARP1's ability to form condensates.

  • Research suggests that caspase-3 cleaves PARP1 between ZnF2 and ZnF3 during apoptosis, potentially disrupting condensate formation .

• Experimental approaches:

  • Combine immunofluorescence using Cleaved-PARP1 (G215) Antibody with analysis of condensate dynamics using live-cell imaging.

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess how cleavage affects the mobility of PARP1 within condensates.

  • Apply super-resolution microscopy to visualize the spatial organization of cleaved vs. uncleaved PARP1 at DNA damage sites.

• Research relevance:

  • Understanding how PARP1 cleavage affects condensate formation may reveal new mechanisms by which apoptosis disrupts DNA repair.

  • This approach could identify novel therapeutic targets at the intersection of phase separation biology and cell death pathways.

This emerging area represents an exciting frontier in PARP1 research where Cleaved-PARP1 (G215) Antibody can provide valuable insights into the structural and functional consequences of PARP1 cleavage beyond its enzymatic inactivation .

What are the latest developments in using cleaved PARP1 detection as a biomarker in pathological conditions?

Cleaved PARP1 detection using specific antibodies is increasingly being explored as a biomarker in various pathological conditions:

• Cancer research applications:

  • Monitoring therapy-induced apoptosis in cancer cells using Cleaved-PARP1 (G215) Antibody provides a direct measure of treatment efficacy.

  • The ratio of cleaved to total PARP1 can predict treatment response in certain cancer types.

  • Cleaved PARP1 detection in circulating tumor cells or extracellular vesicles is being investigated as a minimally invasive biomarker.

• Neurodegenerative diseases:

  • Increased PARP1 cleavage has been observed in neurodegenerative conditions where apoptotic mechanisms contribute to neuronal loss.

  • Cleaved-PARP1 (G215) Antibody can help distinguish between different cell death mechanisms (apoptosis vs. parthanatos) in brain tissue sections.

• Cardiovascular pathologies:

  • Cleaved PARP1 detection in cardiomyocytes following ischemia-reperfusion injury provides insights into cell death mechanisms.

  • The balance between caspase-mediated PARP1 cleavage and PARP1 overactivation may determine cardiomyocyte fate after myocardial infarction.

• Inflammatory disorders:

  • PARP1 cleavage patterns in inflammatory cells can distinguish between resolution phases and chronic inflammation.

  • Monitoring PARP1 cleavage in patient samples may help predict response to anti-inflammatory therapies.

• Technical advancements:

  • Multiplex immunoassays combining Cleaved-PARP1 (G215) Antibody with other cell death markers enhance diagnostic precision.

  • Digital pathology and AI-assisted image analysis are improving the quantification of cleaved PARP1 in tissue samples.

These developments are expanding the utility of Cleaved-PARP1 (G215) Antibody beyond basic research into clinical applications, although further validation is needed before widespread clinical implementation.

What are the most common technical challenges when working with Cleaved-PARP1 (G215) Antibody and how can they be resolved?

Researchers often encounter several technical challenges when working with Cleaved-PARP1 (G215) Antibody. Here are evidence-based solutions to these common problems:

• Weak or absent signal:

  • Possible causes: Insufficient apoptosis induction, degraded antibody, suboptimal antibody concentration, low protein loading.

  • Solutions:

    • Verify apoptosis induction using positive controls (Jurkat cells treated with staurosporine) .

    • Optimize antibody concentration through a dilution series (1:500 to 1:2000) .

    • Increase protein loading (40-60 μg per lane).

    • Extend primary antibody incubation time (overnight at 4°C).

    • Use freshly prepared samples and enhanced chemiluminescence detection.

• High background signal:

  • Possible causes: Insufficient blocking, excessive antibody concentration, inadequate washing.

  • Solutions:

    • Increase blocking time (1-2 hours) or concentration (5% milk/BSA).

    • Optimize antibody dilution.

    • Extend and add wash steps (at least 4 x 5 minutes with TBST).

    • Use freshly prepared buffers.

    • Consider switching blocking agents (BSA vs. milk).

• Multiple or unexpected bands:

  • Possible causes: Cross-reactivity, partial degradation, alternative cleavage products, non-specific binding.

  • Solutions:

    • Include positive controls to identify the correct band (89 kDa).

    • Add protease inhibitors to lysis buffer to prevent degradation.

    • Consider using caspase inhibitors in parallel samples to confirm specificity of the 89 kDa band.

    • Optimize sample preparation and storage conditions.

    • Perform peptide competition assays to confirm specificity.

• Inconsistent results between experiments:

  • Possible causes: Variable apoptosis induction, inconsistent sample preparation, antibody degradation.

  • Solutions:

    • Standardize apoptosis induction protocols.

    • Normalize data to loading controls.

    • Store antibody in small aliquots to avoid freeze-thaw cycles.

    • Maintain consistent incubation times and temperatures.

    • Include positive controls in every experiment.

Following these evidence-based troubleshooting approaches will help researchers obtain reliable and reproducible results when working with Cleaved-PARP1 (G215) Antibody.