PRIMPOL Antibody, FITC conjugated

What is PRIMPOL and why is it important in genomic research?

PRIMPOL (DNA-directed primase/polymerase protein) is a multifunctional enzyme that displays both translesion synthesis (TLS) and DNA repriming capabilities. It plays a crucial role in maintaining genomic stability through its involvement in DNA replication and repair processes. The significance of PRIMPOL lies in its anti-mutagenic activity, particularly in preventing mutations induced by members of the APOBEC/AID family of cytosine deaminases . Studies have demonstrated that PRIMPOL deficiency leads to hypersensitivity to DNA-damaging agents, highlighting its importance in genome maintenance . PRIMPOL's ability to reprime DNA synthesis downstream of lesions on the leading strand makes it an essential component in preventing error-prone translesion synthesis while simultaneously promoting error-free homology-directed repair .

How does FITC conjugation affect antibody performance in PRIMPOL detection?

FITC (Fluorescein isothiocyanate) conjugation provides direct visualization capabilities without requiring secondary antibodies, streamlining immunofluorescence workflows. The FITC-conjugated PRIMPOL antibody has an excitation/emission profile of 499/515nm and is compatible with the 488nm laser line . This conjugation enables direct visualization of PRIMPOL localization within cellular compartments, particularly useful for tracking its dynamic recruitment to chromatin in response to DNA damage . The conjugation process maintains antibody specificity while providing a strong fluorescent signal, though researchers should be aware that extensive exposure to light might lead to photobleaching. To preserve optimal performance, FITC-conjugated antibodies should be stored protected from light, and exposure time during microscopy should be carefully controlled to balance signal intensity with photobleaching concerns .

What are the optimal storage conditions for maintaining PRIMPOL Antibody, FITC conjugated activity?

To maintain the optimal activity of the FITC-conjugated PRIMPOL antibody, it should be stored at -20°C in aliquots to minimize freeze-thaw cycles . The antibody is supplied in a buffer containing 0.01M PBS (pH 7.4), 0.03% Proclin-300 as a preservative, and 50% glycerol to prevent freezing damage . When handling the antibody, it's critical to protect it from light exposure, as FITC is susceptible to photobleaching . For short-term storage during experimental procedures, keep the antibody on ice and protected from light. If long-term storage at -80°C is chosen, ensure complete thawing before use and avoid repeated freeze-thaw cycles, as these can significantly reduce antibody performance and fluorescent signal intensity . Proper storage not only preserves the antibody's immunoreactivity but also maintains the fluorescence intensity of the FITC conjugate.

What are the validated applications for PRIMPOL Antibody, FITC conjugated, and what dilutions are recommended?

The PRIMPOL Antibody, FITC conjugated has been validated for multiple applications with specific recommended dilution ranges for optimal results. For ELISA applications, the recommended dilution range is 1:2000-1:10000, allowing for sensitive detection of the target protein in solution-based assays . For immunohistochemistry (IHC), the antibody performs optimally at dilutions between 1:20-1:200, as demonstrated in paraffin-embedded human placenta tissue samples . In immunofluorescence (IF) applications, dilutions of 1:50-1:200 provide excellent results, as validated in HepG2 cells . The antibody shows strong reactivity with human samples across these applications . For novel applications or cell lines not previously tested, optimization experiments starting with these recommended dilution ranges and including appropriate controls are advisable to determine the optimal antibody concentration for specific experimental conditions.

How can I effectively visualize PRIMPOL recruitment to DNA damage sites using the FITC-conjugated antibody?

To effectively visualize PRIMPOL recruitment to DNA damage sites using FITC-conjugated antibody, implement a DNA damage induction protocol followed by temporal immunofluorescence analysis. Based on research methods, treat cells with interstrand crosslink (ICL)-inducing agents such as mitomycin C (MMC) or UVA-activated trimethyl-psoralen (TMP-UVA) . For localized damage visualization, employ a laser microirradiation approach where TMP-treated cells are irradiated with a UVA laser beam to generate ICLs in defined nuclear regions . Fix cells at specific timepoints post-damage (e.g., 1 hour post-irradiation), permeabilize, and perform immunostaining with the PRIMPOL Antibody, FITC conjugated at 1:50-1:200 dilution . Counter-stain with DNA damage markers like γH2AX and nuclear dyes. For optimal visualization, use confocal microscopy with the appropriate filter sets for FITC (excitation/emission: 499/515nm) . Include PrimPol knockout cells as negative controls to confirm antibody specificity, as research has shown absence of signal in these controls .

What cell fractionation protocol works best for analyzing PRIMPOL chromatin association after DNA damage?

For optimal analysis of PRIMPOL chromatin association following DNA damage, implement a sequential cell fractionation protocol that effectively separates chromatin-bound proteins from soluble nuclear and cytoplasmic fractions. Begin by treating cells with ICL-inducing agents such as MMC (2 μg/ml) or TMP-UVA to promote PRIMPOL recruitment to chromatin . At specific timepoints post-treatment, harvest cells and perform fractionation by first lysing cells in a cytoplasmic extraction buffer (containing detergent but low salt) on ice. Following centrifugation, collect the supernatant as the cytoplasmic fraction and extract the nucleoplasmic fraction from the pellet using nuclear extraction buffer (higher salt concentration). The remaining pellet represents the chromatin-bound fraction, which should be solubilized in a buffer containing nucleases or higher salt concentrations plus sonication to release chromatin-bound proteins. Research has shown that both MMC and TMP-UVA treatments cause accumulation of PrimPol protein specifically on chromatin, coincident with the ubiquitylation and chromatin association of FANCD2, a marker of ICL repair . Always perform parallel immunoblotting for fraction-specific markers (e.g., tubulin for cytoplasm, histone H3 for chromatin) to confirm clean separation.

How can I quantitatively assess PRIMPOL recruitment to chromatin using the FITC-conjugated antibody?

To quantitatively assess PRIMPOL recruitment to chromatin using FITC-conjugated antibody, implement a multi-parameter analysis approach combining fluorescence intensity measurements with spatial colocalization metrics. Begin by acquiring high-resolution Z-stack images of treated and control cells using consistent exposure settings. For analysis, define regions of interest (ROIs) encompassing chromatin areas (using DNA staining) and measure the mean fluorescence intensity (MFI) of PRIMPOL-FITC signal within these regions using image analysis software. Calculate the nuclear/cytoplasmic ratio of PRIMPOL-FITC signal to establish a recruitment index. For micro-irradiation experiments, measure the fluorescence intensity profile across the laser path and calculate fold enrichment compared to non-irradiated nuclear regions . Research has shown that following TMP-UVA treatment, approximately 44% of cells display PRIMPOL recruitment to laser-induced damage paths . For chromatin association studies, combine immunofluorescence with biochemical fractionation approaches, normalizing PRIMPOL-FITC signal to chromatin markers. For statistical validity, analyze at least 100-200 cells per condition across three independent experiments, and apply appropriate statistical tests (ANOVA with post-hoc analysis) to determine significance between treatment groups.

What control experiments are essential when studying PRIMPOL localization in response to DNA damage?

When studying PRIMPOL localization in response to DNA damage using the FITC-conjugated antibody, several critical control experiments must be performed to ensure data validity. First, include a PRIMPOL knockout (KO) cell line as a negative control to confirm antibody specificity, as research has demonstrated absence of signal in these controls . Second, perform untreated controls alongside DNA damage treatments to establish baseline localization patterns. For DNA damage experiments, include a no-damaging agent control (e.g., UVA laser without TMP sensitization) to verify that observed changes result from specific DNA lesions rather than non-specific effects . Additionally, implement positive controls by co-staining for established DNA damage markers such as γH2AX, which should show clear recruitment to damage sites . For FITC signal validation, include an isotype control antibody conjugated to FITC to assess non-specific fluorescence. When studying temporal dynamics, include multiple time points post-damage to capture the full recruitment and resolution profile. Finally, when investigating PRIMPOL's role in specific repair pathways, include knockdowns or inhibitors of key pathway components (e.g., NEIL3, FANCA) to determine pathway dependencies .

How do I interpret differences in PRIMPOL localization patterns between leading and lagging strand replication?

Interpreting differences in PRIMPOL localization patterns between leading and lagging strand replication requires specialized experimental approaches and careful data analysis. First, implement strand-specific labeling using pulse-chase methods with halogenated nucleotides combined with strand-discriminating techniques. When analyzing PRIMPOL-FITC signal distribution, look for asymmetric patterns at replication forks, as research indicates PRIMPOL shows strand-biased anti-mutagenic activity with preferential repriming downstream of AP-sites on the leading strand . For quantitative assessment, measure PRIMPOL-FITC signal intensity along both strands and calculate their ratio to determine strand preference. When interpreting these patterns, it's important to understand that PRIMPOL's leading strand activity helps prevent error-prone translesion synthesis while simultaneously promoting error-free homology-directed repair . The specific enrichment of PRIMPOL on the leading strand correlates with its role in counteracting C>G transversions during somatic hypermutation . For comprehensive interpretation, combine immunofluorescence data with genomic approaches examining mutation spectra in PRIMPOL-deficient versus wild-type cells, which should reveal strand-specific mutational signatures consistent with the observed localization patterns.

How can I resolve weak or diffuse FITC signals when using the PRIMPOL antibody in immunofluorescence?

Weak or diffuse FITC signals when using PRIMPOL Antibody, FITC conjugated can be resolved through systematic optimization of multiple parameters. First, address fixation methods—over-fixation can mask epitopes while under-fixation may disrupt cellular architecture. For PRIMPOL detection, 4% paraformaldehyde fixation for 10-15 minutes typically yields optimal results. Second, evaluate permeabilization conditions, as excessive permeabilization can extract nuclear proteins while insufficient permeabilization limits antibody access; test multiple agents (0.1-0.5% Triton X-100, 0.05-0.1% Saponin) and durations. Third, optimize antibody concentration—for weak signals, adjust concentration to the upper recommended range (1:50 for IF) and extend incubation time to overnight at 4°C. Fourth, enhance signal-to-noise ratio by implementing stringent blocking (5% BSA or 10% serum from the species unrelated to the antibody host) and additional wash steps. For photobleaching concerns, incorporate anti-fade agents in mounting media and minimize exposure during imaging. If these optimization steps fail to improve signal quality, consider signal amplification using anti-FITC antibodies or test alternative fixation methods like methanol-acetone that may better preserve the PRIMPOL epitope. Always verify antibody functionality using positive control samples with known PRIMPOL expression, such as HepG2 cells .

What strategies can address non-specific background when using PRIMPOL Antibody, FITC conjugated?

To address non-specific background when using PRIMPOL Antibody, FITC conjugated, implement a multi-faceted optimization approach targeting each potential source of interference. First, enhance blocking protocols by extending blocking time (1-2 hours) and using a combination of blocking agents (5% BSA with 5-10% serum from a species unrelated to the antibody host) to prevent non-specific binding. Second, optimize antibody dilution through a systematic titration experiment (testing dilutions from 1:20 to 1:200) to identify the concentration that provides optimal signal-to-noise ratio . Third, improve washing protocols by increasing both the number (5-6 washes) and duration (10 minutes each) of wash steps using buffers containing 0.05-0.1% Tween-20. Fourth, examine potential autofluorescence sources by including unstained controls and utilizing spectral imaging if available to distinguish between antibody signal and autofluorescence. For tissues with high autofluorescence, pretreat with sodium borohydride (0.1-1% for 10 minutes) or commercial autofluorescence quenching reagents. Fifth, if nuclear background is problematic, include a pre-extraction step using CSK buffer (Cytoskeletal buffer) with 0.5% Triton X-100 before fixation to remove unbound proteins. Finally, validate specificity by performing parallel staining with PRIMPOL knockout cells as negative controls, which should show absence of specific signal as demonstrated in previous research .

How should I validate the specificity of the PRIMPOL Antibody, FITC conjugated in new experimental models?

To rigorously validate the specificity of PRIMPOL Antibody, FITC conjugated in new experimental models, implement a comprehensive validation protocol incorporating genetic, biochemical, and imaging approaches. First, perform genetic validation using PRIMPOL knockout or knockdown models—research has confirmed antibody specificity through absence of signal in PRIMPOL KO cells . For transient knockdown, use siRNA targeting PRIMPOL and confirm protein reduction via Western blot in parallel with immunofluorescence. Second, conduct peptide competition assays by pre-incubating the antibody with excess recombinant PRIMPOL protein (ideally the immunogen fragment spanning amino acids 305-537) before staining; specific signals should be substantially reduced. Third, validate subcellular localization patterns by comparing PRIMPOL-FITC distribution with published localization data and co-staining with markers of relevant subcellular compartments. Fourth, confirm signal specificity through experimental manipulations—PRIMPOL should show enhanced chromatin recruitment following treatment with DNA damaging agents like MMC or TMP-UVA . Fifth, perform cross-validation using alternate detection methods, such as comparing FITC-conjugated antibody results with those from unconjugated primary antibodies detected with secondary systems. Finally, for absolute confirmation in critical applications, express tagged versions of PRIMPOL (V5-tagged constructs have been validated) and demonstrate co-localization between antibody signal and tag-specific detection.

How can I use PRIMPOL Antibody, FITC conjugated to investigate the interplay between PRIMPOL and the BTR complex in ICL repair?

To investigate the interplay between PRIMPOL and the BTR (BLM-TOP3A-RMI1-RMI2) complex in ICL repair using PRIMPOL Antibody, FITC conjugated, implement a multi-dimensional approach combining co-localization, proximity ligation, and functional analyses. First, establish baseline interaction patterns through co-immunofluorescence of PRIMPOL-FITC with antibodies against BTR components (BLM, TOP3A, RMI1, RMI2) in untreated cells. Then, induce ICLs using MMC or TMP-UVA treatment and track temporal changes in co-localization patterns, as research has demonstrated dynamic interactions between PRIMPOL and BTR components following DNA damage . For higher resolution interaction analysis, perform in situ proximity ligation assays (PLA) between PRIMPOL and individual BTR components before and after DNA damage. To assess functional interdependence, combine immunofluorescence with siRNA-mediated knockdown of BTR components and analyze changes in PRIMPOL recruitment to damage sites. Conversely, examine BTR complex recruitment in PRIMPOL-deficient cells. For mechanistic insights, use chromatin immunoprecipitation (ChIP) with the PRIMPOL antibody followed by re-ChIP for BTR components to identify genomic regions where both complexes co-occupy. Complement these approaches with biochemical co-immunoprecipitation assays, which have previously demonstrated interactions between PRIMPOL and all BTR components, including TOP3A . This comprehensive approach will reveal spatial, temporal, and functional relationships between PRIMPOL and the BTR complex during ICL repair.

What experimental design would best evaluate PRIMPOL's anti-mutagenic activity against APOBEC/AID-induced mutations?

To optimally evaluate PRIMPOL's anti-mutagenic activity against APOBEC/AID-induced mutations, design a comprehensive experimental approach combining cellular, biochemical, and genomic methods. First, establish isogenic cell lines with PRIMPOL knockout, wildtype, and PRIMPOL overexpression, then introduce controlled expression of specific APOBEC/AID family members, particularly APOBEC3B which has established interactions with PRIMPOL . Implement a reporter system containing cytosine-rich sequences within TpC dinucleotides—known APOBEC3B targets —to monitor mutation frequencies. For strand-specific analysis, design constructs allowing discrimination between leading and lagging strand replication to validate PRIMPOL's preferential activity on the leading strand . Complement reporter assays with whole-genome sequencing to catalog mutation spectra, focusing on C>G transversions which PRIMPOL specifically prevents . To assess the mechanistic basis, combine the FITC-conjugated PRIMPOL antibody with EdU or BrdU pulse-labeling to visualize PRIMPOL localization at active replication forks, and correlate this with markers of AP-sites resulting from APOBEC deamination activity. For functional validation, perform rescue experiments with wildtype PRIMPOL and catalytically inactive mutants in PRIMPOL-deficient backgrounds. Finally, extend these studies to primary human breast cancer cells, as research has identified genome-wide anti-mutagenic activity of PRIMPOL against APOBEC3B in invasive breast cancer .

How can PRIMPOL Antibody, FITC conjugated be utilized to investigate the relationship between PRIMPOL and NEIL3 in complementary ICL repair pathways?

To investigate the relationship between PRIMPOL and NEIL3 in complementary ICL repair pathways using PRIMPOL Antibody, FITC conjugated, design a multi-level experimental approach combining visualization, genetic manipulation, and functional readouts. Begin with co-immunofluorescence studies using PRIMPOL-FITC antibody and anti-NEIL3 antibodies to assess potential co-localization or mutually exclusive recruitment patterns at ICL sites induced by TMP-UVA . Implement high-resolution microscopy techniques such as STORM or SIM for nanoscale spatial relationship analysis. Next, establish cell lines with individual or combined knockdown/knockout of PRIMPOL and NEIL3, as research has demonstrated that NEIL3 downregulation further sensitizes PRIMPOL-deficient cells to ICLs, suggesting complementary pathway functions . In these genetic backgrounds, use the FITC-conjugated antibody to track changes in PRIMPOL recruitment dynamics and distribution patterns. For temporal analysis, perform live-cell imaging in cells expressing fluorescently-tagged NEIL3 and treated with the PRIMPOL antibody following micro-irradiation. Complement imaging approaches with functional assays measuring ICL repair efficiency using comet assays or reporter constructs in the various genetic backgrounds. For mechanistic insights, combine genetic approaches with specific ICL-inducing agents that preferentially activate either PRIMPOL or NEIL3-dependent pathways. This comprehensive approach will reveal whether these proteins function sequentially, in parallel, or in competing pathways, expanding upon research showing their complementary roles in processing different types of ICL lesions .

How do I interpret differences in PRIMPOL chromatin association between various DNA damage-inducing treatments?

When interpreting differences in PRIMPOL chromatin association across various DNA damage-inducing treatments, consider both the nature and processing requirements of specific lesions. Using PRIMPOL Antibody, FITC conjugated, quantitatively compare chromatin recruitment following treatment with agents like MMC (producing monoadducts, intrastrand crosslinks, and ICLs) versus TMP-UVA (primarily inducing ICLs) . Research shows both agents cause PRIMPOL accumulation on chromatin coincident with FANCD2 ubiquitylation , but quantitative differences may exist. For proper interpretation, analyze recruitment kinetics by fixing cells at multiple timepoints post-treatment (30min, 1h, 3h, 6h), as different lesions may trigger varying temporal patterns of PRIMPOL association. Consider dose-dependent responses, as recruitment magnitude may correlate with lesion burden. When comparing data, normalize PRIMPOL-FITC signal to appropriate loading controls for each fraction and to measures of damage induction (e.g., γH2AX levels). For comprehensive interpretation, correlate PRIMPOL recruitment patterns with known pathway engagements—for instance, NEIL3-dependent versus NEIL3-independent repair trajectories . Finally, extend analysis to replication-stressed conditions using fork stalling agents like hydroxyurea or aphidicolin, as PRIMPOL functions both in damage bypass and at stalled forks. These comparative analyses will reveal lesion-specific engagement patterns of PRIMPOL and provide mechanistic insights into its context-dependent functions.

What methodologies allow comparison between PRIMPOL's roles in somatic hypermutation versus cancer mutagenesis?

To compare PRIMPOL's roles in somatic hypermutation versus cancer mutagenesis, implement complementary methodologies spanning immunological, cancer, and computational biology approaches. First, establish parallel experimental systems using B-cells undergoing somatic hypermutation and cancer cells (particularly breast cancer models where APOBEC3B-PRIMPOL interactions are established) . In both systems, use PRIMPOL Antibody, FITC conjugated to visualize protein localization during active mutagenesis, combining with markers of the respective processes (activation-induced cytidine deaminase for B-cells; APOBEC3B for cancer cells). Implement strand-specific mutation detection assays, as research shows PRIMPOL specifically counteracts C>G transversions on the leading strand during somatic hypermutation . For genome-wide comparative analysis, perform whole-genome sequencing in PRIMPOL-proficient versus PRIMPOL-deficient backgrounds in both systems, followed by computational extraction of mutational signatures. Apply bioinformatic tools to identify strand-biases and sequence contexts (e.g., TpC motifs in APOBEC3B-mediated mutagenesis) across the genome. For mechanistic comparisons, conduct ChIP-seq using the PRIMPOL antibody in both systems to identify genomic regions where PRIMPOL is recruited. Finally, perform rescue experiments with domain-specific PRIMPOL mutants to determine whether the same functional domains are required for anti-mutagenic activity in both contexts. This comprehensive approach will reveal shared mechanisms and context-specific features of PRIMPOL's anti-mutagenic functions.

How can I use cell synchronization to determine cell cycle-specific dynamics of PRIMPOL localization?

To determine cell cycle-specific dynamics of PRIMPOL localization using PRIMPOL Antibody, FITC conjugated, implement a synchronized cell population approach with multi-parameter imaging analysis. Begin with a double thymidine block protocol as described in the literature—maintain cells in medium with 2.5 mM thymidine for 16h, release for 8h, and block again for synchronization at the G1/S boundary . Following release, collect cells at specific timepoints (2h, 4h, 6h, 8h) representing different S-phase stages and G2. For mitotic enrichment, add nocodazole (50-100 ng/ml) following release. At each timepoint, perform immunofluorescence with PRIMPOL-FITC antibody combined with cell cycle markers (e.g., PCNA patterns for early/mid/late S-phase) and EdU pulse-labeling (10μM, 15-30min) to mark active replication. For DNA damage response studies, synchronize cells, release into S-phase, then treat with DNA damaging agents like MMC before fixation at defined intervals. Implement quantitative image analysis measuring nuclear PRIMPOL-FITC intensity, subcellular distribution patterns, and colocalization with replication markers across the cell cycle. To validate synchronization and cell cycle assignments, perform parallel flow cytometry analysis of DNA content and cyclin expression. For advanced analysis, combine immunofluorescence with proximity ligation assays to detect cell cycle-specific protein interactions between PRIMPOL and known binding partners like the BTR complex components , which show dynamic interactions during cellular responses to DNA damage.