PRIM2 Antibody

What is PRIM2 and why is it important in molecular biology research?

PRIM2 (Primase, DNA, Polypeptide 2) is a 58 kDa regulatory subunit of the DNA primase complex and a component of the DNA polymerase alpha complex, which plays an essential role in DNA replication. It functions by:

• Acting as the regulatory subunit that coordinates with the catalytic subunit (PRIM1) to initiate DNA synthesis

• Synthesizing short RNA primers that are essential for DNA polymerase to elongate DNA strands during replication

• Binding RNA:DNA duplexes and coordinating catalytic activities during primase-to-polymerase switch

PRIM2 is critical for studying DNA replication mechanisms, genome stability, and has emerging significance in cancer research due to its role in cell proliferation .

What applications are PRIM2 antibodies validated for?

PRIM2 antibodies have been validated for multiple experimental techniques including:

Researchers should note that the majority of PRIM2 antibodies show strong validation for Western blotting applications, which remains the primary method for studying PRIM2 expression levels .

What cell lines and tissues show consistent PRIM2 detection?

Based on validation data from multiple sources, PRIM2 antibodies show reliable detection in:

These positive controls are essential when establishing new experimental protocols, as they provide reliable benchmarks for antibody performance .

How should researchers optimize Western blotting protocols for PRIM2 detection?

For optimal PRIM2 detection in Western blotting:

• Sample preparation: Use standard cell lysis buffers with protease inhibitors to prevent degradation

• Protein loading: Load 20-40 μg of total protein per lane for cell lysates

• Gel percentage: Use 10% SDS-PAGE gels for optimal separation around the expected 58-59 kDa molecular weight

• Transfer conditions: Semi-dry or wet transfer (90 minutes at 100V) to PVDF membranes typically yields best results

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Apply at 1:500-1:2000 dilution in 5% BSA/TBST overnight at 4°C

• Detection: Look for bands at approximately 59 kDa, which is the observed molecular weight across multiple validation studies

Note that the observed molecular weight (59 kDa) may differ slightly from the calculated weight, which is common for many proteins due to post-translational modifications .

What controls should be included when validating PRIM2 antibody specificity?

For rigorous validation of PRIM2 antibody specificity, include:

• Positive controls: A375, HeLa, or H460 cell lysates which consistently show PRIM2 expression

• Negative controls:

  • PRIM2 knockdown/knockout samples (siRNA or CRISPR)

  • Secondary antibody-only controls to assess non-specific binding

• Loading controls: Probe for housekeeping proteins (β-actin, GAPDH) to normalize expression

• Peptide competition assays: Pre-incubation with the immunizing peptide should abolish specific signals

• Cross-reactivity assessment: Test in multiple species if cross-reactivity is claimed (human and mouse are most commonly validated)

Published studies have demonstrated antibody validation through knockdown experiments, showing significant reduction in the 58-59 kDa band in PRIM2-silenced cells .

How can researchers effectively use PRIM2 antibodies for immunofluorescence studies?

For successful immunofluorescence experiments with PRIM2 antibody:

• Cell fixation: 4% paraformaldehyde (10 minutes at room temperature) followed by permeabilization with 0.1% Triton X-100

• Blocking: 1-2% BSA in PBS for 30-60 minutes

• Primary antibody: Apply at 1:50-1:500 dilution in blocking buffer overnight at 4°C

• Counterstaining: Include DAPI for nuclear visualization, as PRIM2 shows nucleoplasmic localization

• Controls: Include secondary antibody-only controls

• Expected pattern: PRIM2 typically displays nucleoplasmic staining with potential enrichment at replication foci during S phase

• Validated cell lines: A375 cells have been specifically validated for IF/ICC applications

For co-localization studies, consider pairing with antibodies against other DNA replication proteins such as PCNA, which has been identified as a potential downstream target of PRIM2 .

How can PRIM2 antibodies be utilized to investigate cancer progression mechanisms?

Research has connected PRIM2 with cancer progression through several mechanisms:

• Expression analysis:

  • Use PRIM2 antibodies in tissue microarrays to compare expression across tumor stages

  • Combine with patient outcome data to establish prognostic value

  • Studies have shown PRIM2 expression increases with advanced stages of lung adenocarcinoma

• Functional studies:

  • Employ PRIM2 antibodies to monitor protein levels after genetic manipulation (knockdown/overexpression)

  • Growth inhibition has been observed in lung cancer H1299 cells following PRIM2 knockdown

• Pathway analysis:

  • Use PRIM2 antibodies alongside markers of cell cycle progression and DNA damage

  • PRIM2 has been shown to regulate PCNA expression, influencing DNA replication, mismatch repair, and cell cycle progression

• p53 status correlation:

  • PRIM2 expression appears upregulated when p53 is mutated, suggesting a connection with this key tumor suppressor pathway

ROC curve analysis has demonstrated PRIM2's potential as a diagnostic biomarker for lung cancer, with an area under the curve of 0.904 (95% CI: 0.880–0.927; p < 0.0001) .

What methodologies should researchers employ to investigate PRIM2's role in DNA damage response?

To study PRIM2's involvement in DNA damage response:

• Damage induction and kinetics:

  • Treat cells with DNA damaging agents (UV, cisplatin, hydroxyurea)

  • Use PRIM2 antibodies to track protein levels and localization at different time points post-damage

  • Compare with established DNA damage markers (γH2AX, 53BP1)

• Replication stress:

  • Use DNA combing or DNA fiber assays in conjunction with PRIM2 immunostaining

  • Analyze replication fork progression and stability in PRIM2-depleted cells

• Protein interactions:

  • Perform co-immunoprecipitation with PRIM2 antibodies to identify interaction partners during normal replication versus damage response

  • PCNA has been identified as a potential downstream target regulated by PRIM2

• Senescence assessment:

  • Monitor cellular senescence markers in PRIM2-manipulated cells

  • Research has shown that PRIM2 knockdown causes severe cellular senescence in lung cancer cells

• Chromatin association:

  • Use chromatin fractionation followed by Western blotting with PRIM2 antibodies to assess recruitment to chromatin after DNA damage

These approaches can help delineate PRIM2's specific roles in replication stress responses and DNA repair pathways.

How should researchers interpret discrepancies between expected and observed molecular weights in PRIM2 Western blots?

When encountering differences between calculated and observed molecular weights of PRIM2:

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or other modifications can alter migration patterns

  • Consider phosphatase treatment of samples to assess contribution of phosphorylation

• Isoform expression:

  • Alternative splicing of PRIM2 has been reported, resulting in multiple transcript variants

  • Compare observed bands with transcript data from databases

• Technical considerations:

  • Gel percentage and buffer systems can affect protein migration

  • Use gradient gels (4-15%) to better resolve proteins in the 40-70 kDa range

• Validation approach:

  • Multiple commercial antibodies report an observed molecular weight of approximately 59 kDa despite a calculated weight that may differ

  • Include positive control samples with known PRIM2 expression

  • Perform knockdown/knockout validation to confirm band identity

As noted in product documentation: "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How does PRIM2 relate to other components of the DNA replication machinery?

PRIM2 functions within a complex network of replication proteins:

• DNA polymerase alpha complex:

  • PRIM2 forms a heterodimer with PRIM1 (49 kDa subunit) to create the primase complex

  • This primase complex associates with POLA1 (catalytic subunit) and POLA2 (accessory subunit) to form the complete DNA polymerase alpha complex

• Replication fork recruitment:

  • During S phase, the DNA polymerase alpha complex is recruited to replication forks via interactions with MCM10 and WDHD1

  • PRIM2 coordinates the catalytic activities of PRIM1 and POLA2 during the primase-to-polymerase switch

• Primer synthesis mechanism:

  • Both PRIM1 and PRIM2 are necessary for initial di-nucleotide formation

  • Extension of the primer depends primarily on the catalytic subunit (PRIM1)

  • PRIM2 binds RNA:DNA duplexes, facilitating the handoff between primase and polymerase activities

• Interaction with PCNA:

  • Research has identified proliferating cell nuclear antigen (PCNA) as a potential downstream target of PRIM2

  • PCNA expression is significantly downregulated in PRIM2 knockdown cells

What clinical and prognostic significance does PRIM2 expression have in cancer research?

Emerging research indicates PRIM2 has significant clinical relevance:

• Diagnostic potential:

  • ROC curve analysis shows PRIM2 expression can distinguish lung cancer patients from normal individuals with high accuracy (AUC = 0.904)

  • This suggests potential use as a biomarker for early detection

• Prognostic value:

  • High PRIM2 expression correlates with poor prognosis in lung cancer patients

  • Expression increases progressively with advancing tumor stages

• Therapeutic implications:

  • PRIM2 knockdown inhibits growth of lung cancer cells, suggesting it as a potential therapeutic target

  • Association with p53 mutation status indicates PRIM2 may be particularly relevant in p53-mutant tumors

• Cancer-specific pathways:

  • Gene Set Enrichment Analysis (GSEA) shows PRIM2 may promote lung cancer progression by mediating cell cycle and DNA damage repair processes

  • Correlation with PCNA suggests involvement in multiple proliferation-related pathways

• Specific cancer types:

  • Beyond lung cancer, PRIM2 has been implicated in pancreatic ductal adenocarcinoma progression through interactions with FAM111B

These findings highlight the potential of PRIM2 as both a biomarker and therapeutic target in cancer research.

What are the considerations for analyzing PRIM2 expression data in relation to cell cycle and proliferation studies?

When analyzing PRIM2 expression in cell cycle contexts:

• Cell synchronization:

  • Use methods like double thymidine block or nocodazole treatment to synchronize cells at specific cell cycle phases

  • Analyze PRIM2 levels using the antibody at different time points after release

• Cell cycle marker correlation:

  • Co-analyze PRIM2 with established S-phase markers (PCNA, Cyclin A)

  • Consider flow cytometry with PRIM2 antibody staining combined with DNA content analysis

• Expression quantification:

  • Normalize PRIM2 expression to appropriate housekeeping genes that remain stable across cell cycle phases

  • Use densitometry for Western blots with appropriate statistical analysis (typically ANOVA for multi-timepoint comparisons)

• Proliferation context:

  • PRIM2 knockdown studies have demonstrated growth inhibition in cancer cells

  • PRIM2 regulates PCNA, which is critical for both DNA replication and repair pathways

  • Cellular senescence occurs following PRIM2 knockdown, highlighting its role in preventing cellular aging

• p53 status consideration:

  • Analyze PRIM2 expression in relation to p53 status, as research indicates PRIM2 expression is upregulated in p53-mutated contexts

  • This relationship may influence interpretation of proliferation data in different genetic backgrounds

These considerations ensure proper contextual interpretation of PRIM2 expression data in proliferation and cell cycle studies.

How can researchers troubleshoot non-specific binding or high background when using PRIM2 antibodies?

When encountering high background or non-specific signals:

• Blocking optimization:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature)

  • Add 0.1-0.3% Tween-20 to washing buffers

• Antibody dilution adjustment:

  • Further dilute primary antibody (try 1:2000-1:5000 if using at 1:500-1:1000)

  • Reduce incubation time or switch to 4°C overnight instead of room temperature

  • Prepare antibody solution in fresh blocking buffer

• Sample-specific considerations:

  • For tissue samples, add an additional blocking step with 10% serum from the secondary antibody host species

  • Consider pre-adsorption of antibody with cell/tissue lysates from irrelevant species

• Washing optimization:

  • Increase number and duration of washing steps (5-6 washes, 10 minutes each)

  • Use gentle agitation during washing

• Secondary antibody adjustments:

  • Ensure secondary antibody is highly cross-adsorbed to prevent cross-reactivity

  • Further dilute secondary antibody

  • Consider using polymer detection systems for enhanced specificity

• Validation controls:

  • Always include secondary-only controls

  • Consider using PRIM2 knockdown samples as negative controls to identify non-specific bands

Most PRIM2 antibodies are affinity-purified, which should minimize but not eliminate the potential for non-specific binding .

What approaches should researchers take when adapting PRIM2 antibodies for novel applications or sample types?

When adapting PRIM2 antibodies for new applications:

• Pilot testing strategy:

  • Begin with validated positive controls (A375, HeLa cells)

  • Perform titration experiments with a range of antibody concentrations

  • Include appropriate negative controls (knockdown/knockout samples if available)

• Cross-species applications:

  • Most PRIM2 antibodies are validated for human and mouse samples

  • For other species, perform sequence homology analysis of the immunogen region

  • Begin with Western blotting to confirm antibody reactivity before attempting more complex applications

• New application development:

  • For immunohistochemistry: Test multiple antigen retrieval methods (citrate, EDTA at different pH)

  • For flow cytometry: Compare fixation and permeabilization protocols (paraformaldehyde vs. methanol)

  • For ChIP applications: Optimize crosslinking times and sonication conditions

• Sample-specific modifications:

  • For tissue lysates: Enhance extraction with specialized buffers containing increased detergent concentrations

  • For primary cells: Consider using increased protein amounts to compensate for potentially lower expression

• Validation approach:

  • Whenever possible, confirm results with multiple antibodies targeting different epitopes of PRIM2

  • Use recombinant PRIM2 protein as a positive control for novel applications