PRIMPOL Antibody, Biotin conjugated

What is PRIMPOL and why are PRIMPOL antibodies important in research?

PRIMPOL is an unusual mammalian primase-polymerase belonging to the archaeo-eukaryotic primase superfamily. Unlike conventional primases, PRIMPOL has a preference for dNTPs over NTPs, allowing it to synthesize DNA primers and function as a DNA-dependent DNA polymerase . PRIMPOL antibodies are essential research tools that enable the detection, localization, and functional analysis of this enzyme in various experimental contexts.

PRIMPOL plays critical roles in:

• Reinitiating stalled mtDNA replication

• Repriming DNA synthesis after replication stress

• Facilitating DNA replication restart downstream of DNA lesions

• Promoting hematopoietic stem cell proliferation and bone marrow reconstitution

Antibodies against PRIMPOL allow researchers to study these processes in detail by enabling protein detection in immunoblotting, immunofluorescence, and immunoprecipitation experiments.

How does biotin conjugation enhance PRIMPOL antibody functionality?

Biotin conjugation of PRIMPOL antibodies offers several methodological advantages:

• Enhanced sensitivity: The biotin-streptavidin system provides signal amplification, allowing detection of low-abundance PRIMPOL proteins.

• Versatility: Biotin-conjugated antibodies can be used with various detection systems since streptavidin can be coupled to different reporter molecules.

• Multiplexed detection: When used with streptavidin conjugated to different fluorophores, biotin-labeled PRIMPOL antibodies can be combined with other detection systems for co-localization studies.

• Reduced background: In some experimental systems, biotin-streptavidin detection can provide cleaner results than direct conjugation systems.

Research demonstrates that streptavidin can be effectively used with biotin-conjugated antibodies for PRIMPOL detection . This approach is particularly valuable when studying low-abundance PRIMPOL at replication forks or in specific cellular compartments.

What are the recommended protocols for using biotin-conjugated PRIMPOL antibodies in immunofluorescence?

For optimal immunofluorescence using biotin-conjugated PRIMPOL antibodies:

• Fixation: Use 4% paraformaldehyde (10-15 minutes at room temperature) or methanol (10 minutes at -20°C) depending on the epitope accessibility.

• Permeabilization: Apply 0.2-0.5% Triton X-100 in PBS for 5-10 minutes.

• Blocking: Use 3-5% BSA with 0.1% Tween-20 in PBS for 1 hour to reduce non-specific binding.

• Primary antibody incubation: Apply biotin-conjugated PRIMPOL antibody (typically 1:100-1:500 dilution) for 1-2 hours at room temperature or overnight at 4°C.

• Detection: Incubate with fluorophore-conjugated streptavidin (e.g., Alexa Fluor-tagged streptavidin) at 1:500-1:1000 for 1 hour at room temperature.

• Counterstaining: DAPI (1 μg/ml) for nuclear visualization.

Live cells can also be used with biotin-conjugated antibodies as demonstrated in published protocols . When designing experiments, consider that streptavidin from different manufacturers (such as BD) may have varying sensitivity and specificity profiles.

How can I validate PRIMPOL antibody specificity and functionality before experimental use?

Rigorous validation of PRIMPOL antibodies is essential for experimental reliability:

• Western blot validation:

  • Compare PRIMPOL detection in wild-type vs. PRIMPOL knockout or knockdown cells

  • Verify expected molecular weight (approximately 65 kDa for full-length human PRIMPOL)

  • Test phosphatase treatment for phospho-specific antibodies

• Immunoprecipitation validation:

  • Confirm enrichment of PRIMPOL from cell lysates

  • Verify interaction with known binding partners (e.g., RPA)

• Immunofluorescence validation:

  • Compare staining patterns between wild-type and PRIMPOL-depleted cells

  • Verify expected subcellular localization (nuclear and mitochondrial)

  • Confirm co-localization with replication stress markers after damage induction

• Functional validation:

  • Verify that the antibody does not interfere with PRIMPOL's enzymatic activities

  • For phospho-specific antibodies (like pS255), confirm signal increase after appropriate treatments

Published studies have demonstrated successful antibody generation and validation strategies, including use of lambda phosphatase treatment to confirm phospho-specific antibody binding .

What are the optimal conditions for detecting phosphorylated PRIMPOL using specific antibodies?

Detecting phosphorylated PRIMPOL requires careful experimental design:

• Phospho-specific antibody selection: Custom antibodies targeting specific phosphorylation sites, such as PRIMPOL pS255, have been successfully generated and used in research .

• Lysis buffer composition:

  • Use phosphatase inhibitors (e.g., sodium fluoride, sodium vanadate, PhoSTOP)

  • Include 50 mM Tris (pH 7.4), 200 mM NaCl, 1% Igepal CA-630, 1 mM EDTA

  • Add protease inhibitor cocktail and PMSF (1 mM)

• Sample preparation:

  • Maintain samples at 4°C throughout processing

  • Process samples quickly to minimize dephosphorylation

  • Consider lambda phosphatase treatment as a negative control

• Detection optimization:

  • For immunoblotting, use fresh transfer buffers without methanol

  • Block with BSA rather than milk (phospho-epitopes can bind to casein)

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

Research has demonstrated that CHK1 phosphorylates PRIMPOL at S255 to promote repriming activity during replication stress, making phospho-specific antibodies valuable tools for studying this regulatory mechanism .

How can I design experiments to distinguish between PRIMPOL's primase versus polymerase activities using antibody-based approaches?

Distinguishing between PRIMPOL's dual activities requires careful experimental design:

• Variant-specific antibody approaches:

  • Use antibodies against distinct PRIMPOL domains (e.g., zinc finger primase domain vs. polymerase domain)

  • Compare localization patterns to infer which activity predominates in different contexts

• Functional complementation experiments:

  • Use PRIMPOL variants with specific activity deficiencies:

    • Y89D: Reduced polymerase activity but primase-proficient

    • ZF-KO: Polymerase-proficient but primase-deficient

    • 1-354: Truncated variant lacking primase activity

  • Track protein localization using antibodies during complementation studies

• Activity-specific interaction partners:

  • Use proximity ligation assays with biotin-conjugated PRIMPOL antibodies

  • Identify differences in interaction partners when different activities are required

• Chromatin fractionation:

  • Separate chromatid-bound from soluble PRIMPOL using different extraction conditions

  • Use biotin-conjugated antibodies for detection in different fractions

Published research demonstrates that PRIMPOL's primase activity, rather than its TLS activity, is critical for cellular tolerance to replication stalling lesions . This distinction can be experimentally tested using the approaches outlined above.

Research Applications and Data Analysis

When studying PRIMPOL localization during replication stress:

• Timing considerations:

  • PRIMPOL recruitment is often transient and can be missed if sampling points are inappropriate

  • Include multiple time points after damage induction (e.g., 15 min, 30 min, 1 h, 2 h, 4 h)

  • Consider synchronized cell populations to capture cell cycle-dependent effects

• Damage-specific responses:

  • Different lesions recruit PRIMPOL with varying kinetics and efficiency:

    • Cisplatin: Induces interstrand crosslinks requiring PRIMPOL-mediated bypass

    • Hydroxyurea: Causes nucleotide depletion and replication fork stalling

    • MMS: Generates abasic sites bypassed by PRIMPOL

    • CTNAs: Require PRIMPOL's primase activity for efficient bypass

• Co-localization analysis:

  • Use biotin-conjugated PRIMPOL antibodies with antibodies against:

    • γH2AX (DNA damage marker)

    • PCNA (replication fork marker)

    • RPA (single-stranded DNA marker)

    • Specific repair factors (e.g., RAD51, SMARCAL1, ZRANB3)

• Multi-parameter analysis:

  • Consider both intensity and pattern of PRIMPOL staining

  • Quantify co-localization coefficients with replication/damage markers

  • Track changes in both nuclear and mitochondrial PRIMPOL pools

Research has demonstrated that PRIMPOL's recruitment and function varies substantially depending on the nature of replication stress, with different requirements for primase versus polymerase activity in different contexts .

How can I use PRIMPOL antibodies to investigate mitochondrial DNA maintenance functions?

PRIMPOL plays important roles in mitochondrial DNA maintenance:

• Mitochondrial isolation protocols:

  • Isolate intact mitochondria using differential centrifugation

  • Verify purity using mitochondrial markers (e.g., TOM20, COX4)

  • Use proteinase K treatment to distinguish outer membrane from matrix proteins

• Mitochondrial PRIMPOL detection:

  • Use biotin-conjugated PRIMPOL antibodies with fluorophore-conjugated streptavidin

  • Co-stain with mitochondrial markers (e.g., MitoTracker dyes)

  • Confirm specificity using PRIMPOL knockout controls

• mtDNA replication stress models:

  • Ethidium bromide (low dose): Creates mtDNA replication stress

  • Chain-terminating nucleoside analogs: Stall mtDNA polymerase γ

  • Reactive oxygen species inducers: Generate oxidative mtDNA damage

• Functional readouts:

  • mtDNA copy number (qPCR)

  • mtDNA integrity analysis

  • Mitochondrial transcription levels

  • Respiratory chain function

Research has demonstrated that PRIMPOL can reinitiate stalled mtDNA replication and can prime mtDNA replication from non-conventional origins . The enzyme is specifically required for replication reinitiation after mtDNA damage, highlighting its importance for mitochondrial genome maintenance .

What are common issues with biotin-conjugated antibodies and how can they be addressed?

Several technical challenges may arise when using biotin-conjugated PRIMPOL antibodies:

• High background signal:

  • Cause: Endogenous biotin in biological samples

  • Solution: Pre-block with avidin/streptavidin before adding biotin-conjugated antibody

  • Alternative: Use avidin/biotin blocking kit (Vector Laboratories)

• Reduced sensitivity over time:

  • Cause: Biotinylation site near antibody binding region

  • Solution: Use antibodies with site-specific biotinylation away from antigen binding site

  • Alternative: Store antibodies in single-use aliquots with stabilizing proteins

• Cross-reactivity:

  • Cause: Non-specific binding to similar epitopes

  • Solution: Pre-absorb antibody with related proteins

  • Alternative: Validate with PRIMPOL knockout controls

• Variable signal intensity:

  • Cause: Inconsistent biotin:antibody ratio

  • Solution: Use antibodies with defined biotinylation ratio

  • Alternative: Normalize to internal controls in each experiment

• Biotin interference with PRIMPOL function:

  • Cause: Biotin modification affects protein interactions

  • Solution: Compare results with non-biotinylated antibodies

  • Alternative: Use molecular or genetic approaches to confirm findings

Published protocols demonstrate successful use of streptavidin with biotin-conjugated antibodies for live cell studies , suggesting these challenges can be overcome with proper experimental design.

How can I distinguish between different PRIMPOL phosphorylation states in complex experimental systems?

Analyzing PRIMPOL phosphorylation states requires specialized approaches:

• Phosphorylation-specific antibodies:

  • Use custom antibodies raised against specific phospho-epitopes (e.g., pS255)

  • Validate with lambda phosphatase treatment

  • Include both phosphorylated and non-phosphorylated controls

• Two-dimensional electrophoresis:

  • Separate PRIMPOL by isoelectric point followed by molecular weight

  • Detect with biotin-conjugated PRIMPOL antibodies

  • Compare patterns before and after phosphatase treatment

• Phos-tag gel electrophoresis:

  • Use Phos-tag™ acrylamide to separate phosphorylated forms

  • Detect with general PRIMPOL antibodies

  • Correlate mobility shifts with specific modifications

• Mass spectrometry approaches:

  • Immunoprecipitate PRIMPOL using biotin-conjugated antibodies

  • Analyze phosphorylation sites by mass spectrometry

  • Quantify relative abundance of different phosphoforms

Research has demonstrated that CHK1 phosphorylates PRIMPOL at S255 to promote repriming during replication stress . This phosphorylation can be detected using custom phospho-specific antibodies and is functionally important for cellular resistance to DNA damage .

What strategies can overcome low PRIMPOL detection sensitivity in challenging samples?

For enhanced detection of low-abundance PRIMPOL:

• Signal amplification systems:

  • Tyramide signal amplification (TSA) with biotin-conjugated antibodies

  • Polymer-based detection systems (e.g., EnVision)

  • Rolling circle amplification for extreme sensitivity

• Sample enrichment:

  • Chromatin immunoprecipitation to concentrate DNA-bound PRIMPOL

  • Subcellular fractionation to enrich for nuclear or mitochondrial pools

  • iPOND (isolation of proteins on nascent DNA) to capture replication-associated PRIMPOL

• Specialized imaging:

  • Super-resolution microscopy (STORM, PALM, SIM)

  • Expansion microscopy for physical magnification

  • Proximity ligation assay (PLA) to detect PRIMPOL interactions

• Alternative detection methods:

  • Capillary western blot systems (e.g., Jess, Wes)

  • Ultrasensitive ELISA formats

  • Mass cytometry for single-cell protein detection

Research has employed multiple detection systems with PRIMPOL antibodies, including fluorescent secondary antibodies (Alexa Fluor 488, Alexa Fluor 594), HRP-based detection, and infrared imaging systems (IRDye 800CW, StarBright Blue 700) , demonstrating the versatility of detection approaches.

How can PRIMPOL antibodies be used to investigate the interplay between PRIMPOL and chromatin remodeling factors?

Emerging research suggests connections between PRIMPOL and chromatin regulation:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Use biotin-conjugated PRIMPOL antibodies for ChIP-seq

  • Map PRIMPOL binding sites genome-wide

  • Correlate with chromatin marks and remodeler occupancy

• Proximity-based proteomics:

  • BioID or APEX2 fusions with PRIMPOL

  • Identify chromatin-associated interaction partners

  • Validate interactions with co-immunoprecipitation using biotin-conjugated antibodies

• Chromatin accessibility analysis:

  • Compare chromatin states in wild-type versus PRIMPOL-deficient cells

  • Assess changes in chromatin structure at replication stress sites

  • Correlate PRIMPOL activity with nucleosome positioning

Research has identified connections between PRIMPOL and several chromatin remodeling factors, including HLTF, ZRANB3, and SMARCAL1 . Notably, POLα inhibition leads to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal , suggesting complex interactions between PRIMPOL and chromatin remodeling machinery during replication stress.

What role does PRIMPOL play in specialized cell types, and how can antibody-based approaches investigate this?

PRIMPOL has context-specific functions in different cell types:

• Hematopoietic stem cells (HSCs):

  • PRIMPOL is essential for sustained HSC proliferation and bone marrow reconstitution

  • Use biotin-conjugated antibodies to track PRIMPOL in HSC differentiation

  • Compare PRIMPOL levels and localization across hematopoietic lineages

• B cells and antibody diversification:

  • Studies in DT40 B cells show PRIMPOL may bypass abasic sites during IgV hypermutation

  • Use immunofluorescence to track PRIMPOL during antibody diversification

  • Correlate PRIMPOL activity with mutation patterns

• Neurons and post-mitotic cells:

  • Investigate PRIMPOL's role in non-replicating cells

  • Focus on mitochondrial functions in high-energy demanding tissues

  • Track PRIMPOL localization during neuronal stress responses

• Cancer cells:

  • Compare PRIMPOL levels between normal and transformed cells

  • Correlate PRIMPOL expression with drug resistance profiles

  • Use biotin-conjugated antibodies to assess PRIMPOL as a biomarker

Research demonstrates that PRIMPOL plays critical roles in specific cellular contexts, such as HSC proliferation and bone marrow reconstitution . Further studies using biotin-conjugated PRIMPOL antibodies could elucidate cell type-specific functions and regulatory mechanisms.

How can multi-omics approaches be integrated with PRIMPOL antibody studies to gain comprehensive insights?

Integration of antibody-based studies with multi-omics approaches:

• Proteomics integration:

  • Immunoprecipitate PRIMPOL complexes using biotin-conjugated antibodies

  • Identify interaction partners by mass spectrometry

  • Correlate with proteome-wide changes in PRIMPOL-deficient cells

• Genomics correlations:

  • Combine ChIP-seq using PRIMPOL antibodies with genome-wide screens

  • Correlate PRIMPOL binding sites with genetic vulnerabilities

  • Map PRIMPOL-dependent repriming events genome-wide

• Transcriptomics analysis:

  • Compare gene expression changes in PRIMPOL-deficient versus proficient cells

  • Identify transcriptional consequences of replication stress in PRIMPOL mutants

  • Correlate with PRIMPOL protein levels and localization

• Metabolomics connections:

  • Link PRIMPOL activity to nucleotide metabolism

  • Investigate metabolic signatures of PRIMPOL deficiency

  • Explore connections between mitochondrial PRIMPOL and cellular energetics

Recent CRISPR/Cas9 screens have revealed genetic interactions between PRIMPOL and other DNA repair factors . Integration of these findings with antibody-based studies can provide comprehensive insights into PRIMPOL function within the broader cellular context.