Phospho-POLR2A (S2) Recombinant Monoclonal Antibody

What is Phospho-POLR2A (S2) and why is it important in transcription studies?

Phospho-POLR2A (S2) refers to the phosphorylated form of RNA polymerase II's largest catalytic subunit (POLR2A, also known as RPB1) at the serine 2 position within its C-terminal domain (CTD) heptapeptide repeat sequence (YSPTSPS). This specific phosphorylation plays a crucial role in regulating transcription elongation and mRNA processing mechanisms. The CTD of POLR2A contains multiple repeats of this heptapeptide sequence, and the phosphorylation pattern across these repeats creates a "CTD code" that recruits different factors during transcription progression. Specifically, S2 phosphorylation is associated with productive elongation phases of transcription and facilitates the recruitment of splicing factors and other RNA processing machinery. Using antibodies specific to this phosphorylation state allows researchers to track transcriptionally active polymerase complexes and study elongation dynamics across genes .

What applications have been validated for Phospho-POLR2A (S2) antibodies?

Phospho-POLR2A (S2) antibodies have been rigorously validated across multiple experimental platforms, making them versatile tools for transcription research. According to validation data, these antibodies can be successfully employed in:

These protocols have been tested in multiple cell lines including MCF7, HeLa, C2C12, and C6, demonstrating cross-reactivity across human, mouse, and rat samples .

How can I validate the specificity of a Phospho-POLR2A (S2) antibody?

Validating antibody specificity is essential for reliable experimental outcomes. For Phospho-POLR2A (S2) antibodies, implement the following validation strategy:

• Phosphatase treatment control: Treat a portion of your cell lysate with calf intestinal phosphatase (CIP) to remove phosphorylation. Western blot comparison between treated and untreated samples should show signal reduction in the treated lanes, confirming phospho-specificity. Published data shows successful validation using CIP treatment (20μL/400μL) at 37°C for 1 hour in MCF7, HeLa, C2C12, and C6 cell lines .

• Immunofluorescence co-localization: Perform dual staining with antibodies against total POLR2A and phospho-specific POLR2A (S2). Overlap in nuclear signals with distinct enrichment patterns provides spatial validation of specificity.

• ChIP-sequencing profile analysis: Phospho-POLR2A (S2) should show characteristic enrichment patterns with higher signal in gene bodies compared to promoters, distinguishing it from Phospho-POLR2A (S5) which shows promoter enrichment. Validated antibodies demonstrate specific enrichment patterns at representative gene loci like GAPDH .

• Knockdown/knockout validation: Use siRNA against POLR2A or inhibitors of CTD kinases (like CDK9 inhibitors) to reduce the target signal, confirming specificity of detection.

What are essential controls for experiments using Phospho-POLR2A (S2) antibodies?

Robust experimental design requires appropriate controls to ensure valid interpretation of results. When working with Phospho-POLR2A (S2) antibodies, incorporate these essential controls:

How should I optimize immunofluorescence protocols for Phospho-POLR2A (S2) detection?

Immunofluorescence with Phospho-POLR2A (S2) antibodies requires careful optimization to preserve phosphorylation status and nuclear architecture. Follow these methodological recommendations:

• Fixation protocol: Use freshly prepared 4% paraformaldehyde for 10-15 minutes at room temperature. Overfixation can mask epitopes, while underfixation may cause nuclear material loss.

• Permeabilization: Gentle permeabilization with 0.2% Triton X-100 for 5-10 minutes preserves nuclear structure while allowing antibody access. Harsher detergents may extract nuclear proteins.

• Blocking buffer: Use 3-5% BSA in PBS with 0.1% Tween-20 to reduce background. For neuronal cell lines like PC-12, blocking time may need extension to 1-2 hours.

• Antibody dilution: Optimal dilution for Phospho-POLR2A (S2) antibodies in IF applications is between 1:50 and 1:200. For sensitive detection in PC-12 cells, a 1:100 dilution has been validated with Cy3-conjugated secondary antibodies .

• Nuclear counterstain: DAPI works effectively as a nuclear counterstain and helps visualize nuclear architecture. Phospho-POLR2A (S2) should show punctate nuclear staining in transcriptionally active cells.

• Mounting medium: Use anti-fade mounting medium to prevent photobleaching during imaging and analysis.

What factors affect POLR2A S2 phosphorylation levels in experimental systems?

Understanding factors that influence POLR2A S2 phosphorylation is critical for experimental design and data interpretation. Key modulators include:

• Cell cycle phase: S2 phosphorylation levels fluctuate throughout the cell cycle, with higher levels during S and G2 phases when transcriptional activity increases. Synchronize cells or perform cell cycle analysis in parallel for accurate comparisons.

• Transcriptional inhibitors: CDK9 inhibitors (flavopiridol, DRB) rapidly reduce S2 phosphorylation by blocking the kinase responsible for this modification. These can serve as experimental tools or controls.

• Splicing inhibitors: Interestingly, splicing inhibitors like madrasin can indirectly affect POLR2A levels. Research shows that interfering with splicing machinery can lead to intron retention in POLR2A transcripts, reducing its protein levels and consequently affecting S2 phosphorylation patterns .

• XAB2 levels: XAB2 (XPA-binding protein 2) depletion induces severe splicing defects in POLR2A with significant intron retention, leading to substantial loss of POLR2A at both RNA and protein levels. This subsequently impairs global transcription and can confound phosphorylation studies .

• Cellular stress: Various stressors including DNA damage, oxidative stress, and heat shock can alter the CTD phosphorylation pattern by activating stress-responsive kinases and phosphatases.

How does ChIP-seq with Phospho-POLR2A (S2) antibodies inform our understanding of transcription dynamics?

ChIP-seq using Phospho-POLR2A (S2) antibodies provides genome-wide insights into active transcription and has revolutionized our understanding of transcriptional regulation. This methodology reveals:

• Elongation dynamics: S2 phosphorylation increases as RNA polymerase II proceeds from promoter into gene body, creating a characteristic profile that rises through the gene and often extends beyond the polyadenylation site. This pattern distinguishes actively transcribed genes from paused or inactive genes.

• Correlation with mRNA processing: Peaks of S2 phosphorylation often correlate with exon-intron boundaries and RNA processing sites, reflecting the role of this modification in coupling transcription with mRNA processing.

• Enhancer transcription: Low-level S2 phosphorylation at enhancers indicates enhancer RNA (eRNA) transcription, helping identify active enhancer elements genome-wide.

• Protocol optimization: For optimal results, use 5μg antibody for 10-15μg of cross-linked chromatin. Following ChIP, construct sequencing libraries using standard protocols with appropriate size selection (150-300bp fragments) .

• Bioinformatic analysis: Analyze ChIP-seq data using peak-calling algorithms optimized for broad peaks rather than sharp transcription factor binding sites. Normalize to input and compare with total POLR2A profiles to distinguish changes in phosphorylation from changes in occupancy.

ChIP-seq experiments with Phospho-POLR2A (S2) antibodies can identify enrichment patterns at specific genomic regions, as demonstrated in studies showing characteristic profiles at representative gene loci like GAPDH .

What are the advantages of CUT&Tag over ChIP-seq for studying POLR2A S2 phosphorylation?

CUT&Tag (Cleavage Under Targets and Tagmentation) represents a significant methodological advancement for epigenomic profiling, offering several advantages over traditional ChIP-seq for studying POLR2A S2 phosphorylation:

• Higher sensitivity: CUT&Tag requires significantly fewer cells (as low as 10^5 cells with 1μg of Phospho-POLR2A (S2) antibody) compared to ChIP-seq, making it suitable for rare cell populations or limited samples .

• Improved signal-to-noise ratio: CUT&Tag typically yields cleaner data with lower background, as the tagmentation reaction occurs only at antibody-bound sites within the native chromatin environment.

• Preserved nuclear architecture: The method uses intact cells/nuclei rather than sonicated chromatin, potentially preserving higher-order chromatin structures relevant to transcription regulation.

• Faster protocol: CUT&Tag can be completed in 1-2 days versus 3-4 days for ChIP-seq, with fewer washing steps and handling losses.

• Recommended protocol: Successful CUT&Tag has been performed using the CUT&Tag Assay Kit (pAG-Tn5) from 10^5 HeLa cells with 1μg Phospho-POLR2A CTD-S2 Rabbit mAb, along with a secondary Goat Anti-Rabbit IgG(H+L). This approach revealed characteristic enrichment patterns at representative gene loci like GAPDH .

When deciding between methods, consider sample availability, required resolution, and compatibility with downstream analyses. For limited samples or higher resolution needs, CUT&Tag offers advantages, while ChIP-seq remains valuable for cross-comparison with existing datasets.

How does XAB2 depletion specifically affect POLR2A and what mechanisms underlie this relationship?

Research has uncovered a critical relationship between XAB2 (XPA-binding protein 2) and POLR2A expression that impacts global transcription. The mechanistic details include:

• Splicing regulation: XAB2 depletion leads to severe splicing defects in POLR2A transcripts, characterized by significant intron retention. This splicing disruption results in substantial loss of POLR2A at both RNA and protein levels, subsequently impairing global transcription .

• mRNA surveillance pathway involvement: Proteomics analysis after XAB2 depletion revealed upregulation of proteins involved in mRNA surveillance, including Dom34 (PELO). Knockdown of Dom34 partially rescues POLR2A expression by stabilizing its mRNA, suggesting that aberrantly spliced POLR2A transcripts are targeted for degradation by mRNA surveillance mechanisms .

• Spliceosome component interactions: Immunoprecipitation experiments confirmed that XAB2 associates with spliceosome components critical for POLR2A expression. Depletion of specific factors including SNRNP200, EFTUD2, SNW1, PRPF8, PLRG1, and AQR reduces POLR2A expression, indicating a specialized splicing machinery requirement .

• Domain mapping insights: The TPR motifs 2-4 and 11 of XAB2 are particularly important for POLR2A expression, functioning through interaction with SNW1. This structural information provides mechanistic insight into how XAB2 regulates POLR2A processing .

• Cellular senescence connection: XAB2 or POLR2A depletion induces cellular senescence by upregulating p53 and p21. Re-expression of POLR2A after XAB2 depletion alleviates cellular senescence, positioning POLR2A as a major mediator of senescence induced by XAB2 deficiency .

This complex regulatory relationship highlights how splicing defects in a single critical gene (POLR2A) can profoundly impact global transcription and cellular aging pathways.

What is the relationship between POLR2A S2 phosphorylation and cellular senescence?

POLR2A S2 phosphorylation has emerged as a critical regulator in cellular senescence pathways through several interconnected mechanisms:

• Global transcription maintenance: POLR2A S2 phosphorylation is essential for productive transcription elongation. Research demonstrates that disruption of POLR2A expression or function leads to impaired global transcription, triggering stress responses that culminate in cellular senescence .

• p53-p21 pathway activation: Depletion of POLR2A induces significant cellular senescence markers, with approximately 49.7% of cells showing positive SA-β-gal staining compared to control levels. This senescence phenotype correlates with upregulation of p53 and p21, key mediators of cell cycle arrest and senescence .

• Cell cycle effects: POLR2A deficiency results in cell cycle arrest predominantly at the G2/M phase and inhibits cell proliferation, characteristic features of senescent cells .

• Rescue experiments: Importantly, re-expression of POLR2A after depletion of upstream regulators like XAB2 alleviates cellular senescence, confirming POLR2A's central role in this process rather than being a secondary effect .

• Transcription-coupled stress signaling: Disruption of POLR2A function may trigger transcription-coupled stress signaling pathways that activate p53, potentially through R-loop formation or other transcriptional stress mechanisms.

These findings position POLR2A and its phosphorylation state as central regulators in the cellular senescence program, linking transcriptional regulation directly to cellular aging pathways. This connection has significant implications for understanding age-related pathologies and potential therapeutic interventions.

How do alterations in POLR2A S2 phosphorylation patterns contribute to disease states?

Dysregulation of POLR2A S2 phosphorylation has been implicated in various pathological conditions through disruption of transcriptional regulation:

• Cancer biology: Alterations in POLR2A phosphorylation patterns can drive aberrant gene expression programs in cancer cells. Cancer-specific transcriptional dependencies may emerge from changes in CTD phosphorylation, creating potential therapeutic vulnerabilities. Research confirms that dysregulation of POLR2A phosphorylation has been implicated in various diseases, including cancer .

• Neurological disorders: Proper POLR2A function and phosphorylation is critical for neuronal gene expression programs. Disruption of POLR2A regulation has been associated with neurological conditions, highlighting its importance in neural development and function .

• Senescence-related pathologies: Given POLR2A's role in cellular senescence pathways, dysregulation may contribute to age-related diseases. Research shows that XAB2 depletion causes POLR2A reduction and induces cellular senescence through p53/p21 activation, potentially connecting to age-related tissue dysfunction .

• Developmental disorders: As a master regulator of transcription, POLR2A phosphorylation abnormalities may underlie certain developmental disorders through global perturbation of gene expression programs.

• Stress response pathways: POLR2A phosphorylation serves as an integration point for cellular stress signals. Chronic dysregulation may contribute to stress-related pathologies through persistent activation or inhibition of stress-responsive genes.

Understanding these connections offers potential therapeutic opportunities, including the development of compounds that modulate CTD phosphorylation or target specific transcriptional dependencies in disease states.

What are the most common technical challenges when working with Phospho-POLR2A (S2) antibodies?

Researchers face several technical challenges when working with Phospho-POLR2A (S2) antibodies that can impact experimental outcomes:

How can I optimize Western blot protocols for detecting Phospho-POLR2A (S2)?

Western blot detection of Phospho-POLR2A (S2) requires careful optimization due to the high molecular weight and phosphorylation-specific nature of the target. Follow these methodological recommendations:

• Sample preparation: Lyse cells directly in SDS sample buffer containing phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate, 10mM β-glycerophosphate) to immediately preserve phosphorylation status.

• Gel selection: Use 6-8% polyacrylamide gels to properly resolve the ~270kDa POLR2A protein. Commercial gradient gels (4-15%) can also work well for this high molecular weight protein .

• Transfer conditions: Perform wet transfer at low voltage (30V) overnight at 4°C to ensure complete transfer of high molecular weight proteins. Use 0.45μm PVDF membrane rather than 0.2μm for better retention of large proteins.

• Blocking conditions: Block membranes with 3% BSA in TBST rather than milk, as milk contains phosphatases that can remove the phosphorylation being detected .

• Antibody dilution: Optimal results have been achieved with 1:1000 dilution of primary antibody and 1:10000 dilution of HRP-conjugated secondary antibody (goat anti-rabbit IgG). Incubate primary antibody overnight at 4°C for best results .

• Detection system: Use ECL detection systems with appropriate sensitivity. For weak signals, enhanced chemiluminescence substrates or longer exposure times may be necessary. Published protocols have successfully used ECL Basic Kit with 10-second exposure time .

• Controls: Always include a phosphatase-treated control sample (using CIP treatment: 20μL/400μL at 37°C for 1 hour) to confirm phospho-specificity of detection .

What methodological approaches can resolve contradictory data in POLR2A phosphorylation studies?

When faced with contradictory results in POLR2A phosphorylation studies, employ these methodological approaches to resolve discrepancies: