POLA2 Antibody

What is POLA2 and what is its biological significance in research?

POLA2 (polymerase DNA directed, alpha 2) is the 70 kDa accessory subunit of the DNA polymerase α (polα)/primase complex, which plays a crucial role in DNA replication, particularly during the initiation phase. POLA2 consists of 598 amino acids, with incorporation into the enzyme complex facilitated by its 250 amino acid N-terminal domain . The protein is essential for multiple cellular processes:

• Initiation of DNA replication

• Telomere C-strand fill-in

• DNA double-strand break repair

• Maintenance of gene stability

Recent research has implicated POLA2 in cancer progression, particularly hepatocellular carcinoma, and in rare genetic disorders like telomere biology disorders with Coats plus features . This makes POLA2 antibodies valuable tools for both basic research and clinical investigations.

What types of POLA2 antibodies are available for research applications?

Several types of POLA2 antibodies are available, each with specific characteristics suitable for different research applications:

These antibodies are available in various formats, including:

• Unconjugated primary antibodies

• HRP-conjugated for direct detection

• Fluorescent-conjugated (FITC, PE, Alexa Fluor) for immunofluorescence

• Agarose-conjugated for immunoprecipitation

Selection should be based on the specific experimental requirements, target species, and application.

What are the recommended applications and dilutions for POLA2 antibodies?

POLA2 antibodies have been validated for multiple research applications, each requiring specific dilutions:

Methodological consideration: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" as noted in manufacturer protocols . Cell/tissue-specific optimization may be necessary for robust and reproducible results.

How should POLA2 antibodies be stored and handled for optimal performance?

Proper storage and handling are critical for maintaining antibody activity and specificity:

For antibodies containing BSA (e.g., 20μL sizes with 0.1% BSA), aliquoting may be unnecessary for -20°C storage as indicated by Proteintech . Always check manufacturer's specific recommendations as formulations may vary between suppliers.

How can POLA2 antibodies be utilized to investigate DNA replication mechanisms?

POLA2 antibodies provide powerful tools for studying DNA replication through multiple experimental approaches:

Chromatin Immunoprecipitation (ChIP):

• Immunoprecipitate POLA2-bound DNA to identify replication origins

• Monitor temporal recruitment of POLA2 to replication forks

• Analyze association with specific genomic regions during S phase

Co-immunoprecipitation studies:

• Isolate the polα/primase complex using POLA2 antibodies

• Identify novel interacting partners within the replisome

• Investigate how complex composition changes during replication stress

Immunofluorescence microscopy:

• Track POLA2 localization throughout cell cycle phases

• Co-localize with other replication factors (PCNA, MCM proteins)

• Quantify replication foci formation and dynamics

Functional studies:

• Combine with POLA2 depletion (siRNA/shRNA as used in ) to assess replication defects

• Monitor DNA synthesis using EdU or BrdU incorporation after POLA2 manipulation

• Investigate replication fork stability and restart after genotoxic stress

These approaches can reveal critical insights into how POLA2 contributes to normal replication and how its dysfunction may lead to genomic instability.

What is the role of POLA2 in telomere biology and Coats plus syndrome research?

POLA2, as part of the DNA polymerase α/primase complex, is crucial for telomere C-strand fill-in. Recent research has identified biallelic POLA2 variants in patients with telomere biology disorders (TBDs) with Coats plus features .

Key research findings:

• Biallelic deleterious POLA2 variants were identified in five individuals from two unrelated families

• All affected individuals displayed abnormally short telomeres

• Clinical phenotypes included retinal and gastrointestinal telangiectasias

• These features are consistent with previously described Coats plus syndrome associated with CTC1, STN1, and POT1 mutations

Research applications using POLA2 antibodies:

• Analyze POLA2 recruitment to telomeres using ChIP or IF-FISH

• Investigate interactions with the CST complex (CTC1, STN1, TEN1)

• Compare telomere-bound POLA2 in normal versus patient-derived cells

• Evaluate C-strand fill-in efficiency in the presence of POLA2 variants

This research establishes POLA2 as a novel autosomal recessive gene for TBDs, expanding our understanding of the molecular basis of Coats plus syndrome and related disorders.

How does POLA2 overexpression influence hepatocellular carcinoma progression and immune infiltration?

Recent research has revealed important connections between POLA2, hepatocellular carcinoma (HCC) progression, and immune cell infiltration:

Key research findings from TCGA-LIHC database and HCC patient samples:

Experimental evidence on POLA2 function in HCC:

• Gene knockdown revealed that POLA2 promotes proliferation, migration, invasion, and cell cycle progression in HCC cell lines (SMMC-7721 and HepG2)

• In xenograft models, POLA2 knockdown led to tumor size inhibition, reduced proliferation, and increased necrosis

Relationship with immune microenvironment:

• Significant correlation with tumor-associated macrophage infiltration

• Positive co-expression with immune checkpoints (CD274/PD-L1, CTLA-4, HAVCR2, PDCD1, PDCD1LG2, TIGIT, and LAG3)

• Functional enrichment analysis revealed POLA2 co-expressed genes are linked to immune response regulation

Regulatory mechanism:

• E2F1 transcription factor appears to regulate POLA2 expression in HCC

• This suggests a potential pathway connecting cell cycle regulation and immune infiltration through POLA2

These findings position POLA2 as a potential prognostic marker and therapeutic target in HCC, with POLA2 antibodies serving as valuable tools for investigating these associations.

What methodological considerations are important when using POLA2 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with POLA2 antibodies requires careful attention to methodology to preserve protein interactions while minimizing artifacts:

Antibody selection:

• For POLA2 Co-IP, consider using agarose-conjugated antibodies (e.g., sc-398255 AC )

• Alternatively, use protein A/G beads with unconjugated antibodies (typically 1-5 μg per reaction)

• Validate IP efficiency by Western blot before proceeding to interaction studies

Optimized lysis conditions:

• Use non-denaturing buffers containing 0.1-0.5% mild detergents (NP-40 or Triton X-100)

• Include protease and phosphatase inhibitors to preserve native protein states

• For nuclear proteins like POLA2, ensure efficient nuclear extraction

Protocol refinements:

• Pre-clear lysates with control IgG/beads to reduce non-specific binding

• Include appropriate negative controls (e.g., IgG matched to the host species of your POLA2 antibody)

• For weakly interacting partners, consider crosslinking approaches (e.g., DSP, formaldehyde)

Analysis workflow:

• Perform POLA2 immunoprecipitation from cellular extracts

• Wash thoroughly (3-5 times) with decreasing detergent concentrations

• Elute bound proteins and analyze by Western blot or mass spectrometry

• Confirm results with reciprocal IP or orthogonal interaction methods

When investigating the DNA polymerase α complex, consider that interactions between POLA2 and other subunits (POLA1, PRIM1, PRIM2) may vary during cell cycle phases or under replication stress conditions.

How can researchers validate the specificity of POLA2 antibodies in their experimental systems?

Rigorous validation of POLA2 antibodies is essential for ensuring reliable research results:

Genetic validation approaches:

• RNA interference validation:

  • Transfect cells with POLA2-targeting siRNA/shRNA

  • Compare antibody signal between control and POLA2-depleted samples

  • A specific antibody should show significant signal reduction after knockdown

  • This approach is employed by the Human Protein Atlas for enhanced validation

• Recombinant expression validation:

  • Express tagged POLA2 in cells with low endogenous expression

  • Verify co-localization of antibody signal with the tagged protein

  • Confirm the specificity by Western blot showing the expected molecular weight (68-70 kDa)

Technical validation strategies:

• Multiple independent antibodies:

  • Use at least two antibodies targeting different POLA2 epitopes

  • Compare staining patterns or Western blot results

  • Consistent results increase confidence in specificity

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide or recombinant POLA2

  • Apply pre-absorbed antibody to samples

  • Specific signal should be significantly reduced or eliminated

• Tissue/cell type controls:

  • Include samples with known POLA2 expression levels (e.g., HepG2, COLO 320, HeLa, and MCF-7 cells show positive Western blot signal )

  • Use tissues/cells with differential expression as positive and negative controls

Documentation requirements:

• Record complete antibody information (manufacturer, catalog number, RRID)

• Document all validation experiments performed

• Report observed molecular weight (expected: 68-70 kDa )

• Include all validation data when publishing research using these antibodies

Following these validation practices ensures confidence in experimental results and facilitates reproducibility in POLA2 research.

What approaches are recommended for studying POLA2 post-translational modifications?

Post-translational modifications (PTMs) of POLA2, such as phosphorylation during G2/M phase , can regulate its function, localization, and interactions:

Identification strategies:

• Immunoprecipitation-Mass Spectrometry approach:

  • Immunoprecipitate POLA2 using validated antibodies

  • Analyze by LC-MS/MS to identify modifications and their sites

  • Compare PTM profiles across different cellular conditions

• PTM-specific antibody detection:

  • Use general PTM antibodies (anti-phospho-Ser/Thr, anti-ubiquitin) after POLA2 immunoprecipitation

  • If available, use modification-specific POLA2 antibodies

  • Detect by Western blot using enhanced chemiluminescence or fluorescent detection

Functional analysis methods:

• Cell cycle synchronization studies:

  • Synchronize cells at different cell cycle phases (thymidine block, nocodazole, etc.)

  • Immunoprecipitate POLA2 and analyze PTM status

  • Correlate with DNA polymerase activity and complex formation

• Mutagenesis approaches:

  • Generate non-modifiable POLA2 mutants (e.g., S→A for phosphorylation sites)

  • Express in cells and assess impact on localization and function

  • Compare with phosphomimetic mutations (e.g., S→D/E)

Technical considerations:

• Sample preparation:

  • Include appropriate PTM-preserving inhibitors in lysis buffers:

    • Phosphatase inhibitors (sodium orthovanadate, sodium fluoride)

    • Deubiquitinase inhibitors (N-ethylmaleimide)

    • Protease inhibitors (PMSF, leupeptin, aprotinin)

• Enhanced detection methods:

  • Use Phos-tag SDS-PAGE for improved separation of phosphorylated proteins

  • Employ 2D gel electrophoresis to separate different PTM forms

  • Consider super-resolution microscopy for co-localization of POLA2 with PTM markers

Understanding POLA2 PTMs can provide insights into its regulation during normal DNA replication and how dysregulation may contribute to disease states like cancer or telomere biology disorders.

Future research directions for POLA2 antibody applications

The continued development and application of POLA2 antibodies promise to advance our understanding in several key areas:

• Precision medicine applications:

  • Development of companion diagnostics for HCC based on POLA2 expression

  • Stratification of cancer patients for potential targeted therapies

  • Screening for telomere biology disorders with POLA2 involvement

• Advanced research techniques:

  • Single-cell analysis of POLA2 expression in heterogeneous tumor samples

  • Spatial proteomics to map POLA2 localization within replication complexes

  • High-throughput screens for compounds affecting POLA2 expression or function

• Methodological improvements:

  • Development of more specific monoclonal antibodies against distinct POLA2 epitopes

  • Generation of modification-specific antibodies (phospho-POLA2, etc.)

  • Improved protocols for detecting low-abundance POLA2 complexes

As research advances, POLA2 antibodies will remain essential tools for exploring fundamental biological processes and translating these insights into clinical applications.

Resources for researchers working with POLA2 antibodies

Researchers exploring POLA2 functions can consult several key resources: