POL2A Antibody

What is POLR2A and why is it an important research target?

POLR2A (DNA-directed RNA Polymerase II subunit RPB1) is the largest subunit of RNA polymerase II, the catalytic mechanism responsible for the synthesis of mRNA. The protein is approximately 217 kDa (calculated) with an observed molecular weight of 210-250 kDa on Western blots. It contains an N-terminus with six RNA polymerase domains (amino acids 15-1079) and a C-terminus (CTD) with 52 seven-amino acid repeats (YSPTSPS) that serve as a platform for polymerase subunit interaction . This CTD undergoes phosphorylation during transcription, with different phosphorylation states associated with various stages of the transcription cycle.

POLR2A is pivotal for research because its phosphorylation status directly correlates with transcriptional activity. When hyperphosphorylated, the subunit is called RNA pol IIo; when hypophosphorylated, it becomes RNA pol IIa . These modifications play crucial roles in transcript initiation, elongation, and termination, making POLR2A antibodies essential tools for studying transcription regulation mechanisms.

How do I choose between phospho-specific and total POLR2A antibodies for my research?

The selection between phospho-specific and total POLR2A antibodies should be based on your research question:

Total POLR2A antibodies:

Phospho-specific antibodies:

• Select Ser2 phosphorylation (pSer2) antibodies when studying transcription elongation, as Ser2 phosphorylation occurs predominantly during elongation phase

• Choose Ser5 phosphorylation (pSer5) antibodies when investigating transcription initiation and early elongation, as Ser5 phosphorylation facilitates 5' capping and recruitment of the capping enzyme

• Consider using both pSer2 and pSer5 antibodies for comprehensive analysis of the transcription cycle

For experiments examining transcriptional dynamics, a combination approach using both total and phospho-specific antibodies provides the most comprehensive data . Western blot validation experiments show that phospho-specific antibodies are sensitive to phosphatase treatment, confirming their specificity, as demonstrated in HeLa cell lysates tested with the NB100-1805 antibody .

What validation methods should I use to confirm POLR2A antibody specificity?

A rigorous POLR2A antibody validation protocol should include multiple approaches:

Essential validation methods:

• Western blot with phosphatase treatment: Compare untreated samples with phosphatase-treated samples to confirm phospho-specificity

• Peptide competition assays: Pre-incubate antibodies with phosphorylated and non-phosphorylated peptides to verify epitope specificity

• siRNA knockdown: Analyze samples from cells transfected with POLR2A siRNA alongside untransfected controls

• Cross-reactivity testing: Verify reactivity across intended species (human, mouse, etc.)

The most convincing validation combines multiple approaches. For instance, research data shows that the RNA Polymerase II pSer2 antibody (NB100-1805) specificity can be confirmed by peptide competition assays, demonstrating that pre-incubation with the phosphorylated S2 peptide blocks antibody binding, while pre-incubation with non-phosphorylated peptide or phosphorylated S5 peptide does not affect binding .

How should I optimize Western blot protocols for POLR2A detection?

Optimizing Western blot protocols for POLR2A requires special considerations due to its high molecular weight and various phosphorylation states:

Recommended Western blot protocol for POLR2A:

• Sample preparation:

  • Use NETN buffer or specialized high molecular weight protein extraction buffers

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Load 25-50 μg of nuclear extracts or whole cell lysates

• Gel preparation and running:

  • Use low percentage (6-8%) gels or gradient gels (4-15%) to properly resolve the 210-250 kDa protein

  • Run gels at lower voltage (80-100V) for better resolution of high molecular weight proteins

• Transfer conditions:

  • Use wet transfer methods with 0.45 μm PVDF membranes

  • Transfer at 30V overnight at 4°C for high molecular weight proteins

  • Add 0.1% SDS to transfer buffer to facilitate high molecular weight protein transfer

• Antibody dilutions:

  • Primary antibody: Use 1:500-1:2000 for total POLR2A (e.g., 20655-1-AP)

  • For phospho-specific antibodies: Use 1:2000-1:10000 (e.g., NB100-1805)

  • Secondary antibody: Use HRP-conjugated anti-rabbit or anti-sheep IgG at 1:5000-1:10000

• Detection:

  • Employ ECL-based chemiluminescence with exposure times starting at 3 seconds

  • For weaker signals, consider using enhanced chemiluminescence substrates

This protocol has been validated with multiple POLR2A antibodies including AF6160, 20655-1-AP, and NB100-1805 across various cell lines including HeLa, HepG2, MCF-7, and NIH/3T3 .

What are the best approaches for using POLR2A antibodies in ChIP and ChIP-seq experiments?

POLR2A antibodies are frequently used in ChIP and ChIP-seq experiments to monitor transcriptional activity genome-wide. Here's a methodological approach for optimizing these experiments:

ChIP protocol optimization for POLR2A:

• Antibody selection:

  • For identifying actively transcribing genes: Use phospho-Ser2 antibodies

  • For identifying genes with initiated but paused transcription: Use phospho-Ser5 antibodies

  • For total Pol II occupancy: Use antibodies targeting non-phosphorylated regions

• Chromatin preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Sonicate chromatin to generate fragments of 200-500 bp

  • Use 1-10 million cells per ChIP reaction

• Antibody titration:

  • Test a range of antibody amounts (1, 2, 5, and 10 μg per ChIP) to determine optimal antibody-to-chromatin ratio

  • Include IgG controls (2 μg/IP) as negative controls

• qPCR validation:

  • Design primers for positive controls (constitutively expressed genes like GAPDH and ACTB)

  • Include negative controls (inactive genes like myoglobin or satellite repeats)

  • Calculate recovery as percentage of input

• ChIP-seq considerations:

  • Sequence to a depth of at least 20 million uniquely mapped reads

  • Use peak-calling algorithms designed for broad peaks (typical of Pol II binding)

  • Normalize to input DNA and IgG controls

Experimental data shows that the optimal antibody amount varies by application. For ChIP-qPCR using the C.15200005 antibody, titration experiments demonstrated that 5 μg of antibody per ChIP provided optimal enrichment of Pol II pSer2 at active genes, with 30-40% recovery at the GAPDH locus compared to <5% at inactive genomic regions .

How can I use POLR2A antibodies to study transcription dynamics in fixed tissues and cells?

POLR2A antibodies can be powerful tools for studying transcription in fixed specimens through immunohistochemistry (IHC) and immunofluorescence (IF). Here's a methodological approach:

Protocol for POLR2A detection in fixed specimens:

• Sample preparation:

  • For paraffin-embedded tissues: Use citrate buffer (pH 6.0) for antigen retrieval

  • For frozen sections: Fix with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100

• Antibody selection and dilution:

  • For IHC-paraffin: Use 1:50-1:500 dilution of POLR2A antibodies (e.g., 20655-1-AP)

  • For phospho-specific detection: Use 1:200-1:1000 dilution (e.g., NB100-1805)

• Signal detection options:

  • Chromogenic detection: Use DAB substrate for standard bright-field microscopy

  • Fluorescent detection: Use fluorophore-conjugated secondary antibodies for co-localization studies

• Counterstaining:

  • Nuclear counterstain (DAPI or hematoxylin) to visualize nuclear localization of POLR2A

  • Consider co-staining with markers of specific cell types or other transcription factors

• Controls and validation:

  • Include phosphatase-treated sections as negative controls for phospho-specific antibodies

  • Use tissue/cells known to have high transcriptional activity as positive controls

This approach has been validated in mouse teratoma sections using the NB100-1805 antibody at 1:200 dilution with DAB detection and in human pancreatic cancer tissues using the 20655-1-AP antibody .

Why might I observe multiple bands or variable molecular weights with POLR2A antibodies in Western blots?

Multiple bands or variable molecular weights when detecting POLR2A can be attributed to several biological and technical factors:

Biological factors:

• Phosphorylation states: The hyperphosphorylated form (Pol IIo) runs at a higher molecular weight (~250 kDa) than the hypophosphorylated form (Pol IIa, ~220 kDa)

• CTD length variation: The C-terminal domain can vary in apparent size due to different degrees of phosphorylation across the 52 heptapeptide repeats

• Proteolytic processing: Partial degradation during sample preparation can generate truncated forms

Technical factors:

• Gel percentage: Lower percentage gels (6-8%) resolve the high molecular weight bands better than higher percentage gels

• Sample preparation: Incomplete denaturation or phosphatase activity during extraction can alter band patterns

• Antibody specificity: Some antibodies may recognize specific phosphorylation states or epitopes that are variably accessible

Interpretation guide:

To confirm band identity, compare phosphorylated and dephosphorylated samples. In validation studies with the NB100-1805 antibody, phosphatase treatment abolished the signal, confirming that the antibody specifically recognizes the phosphorylated form of the protein .

How do I resolve inconsistent results between phospho-specific and total POLR2A antibodies?

Inconsistencies between phospho-specific and total POLR2A antibody results often reflect biological realities rather than technical issues. Here's a systematic approach to understanding and resolving these discrepancies:

Common scenarios and interpretations:

• High phospho-signal with low total POLR2A:

  • Interpretation: Increased phosphorylation per molecule rather than increased total protein

  • Validation: Normalize phospho-signal to total POLR2A rather than to loading controls

• Unchanged phospho-signal with increased total POLR2A:

  • Interpretation: More polymerase molecules, but not more active transcription

  • Validation: Examine transcripts of target genes to confirm transcriptional output

• Different localization patterns in IF/IHC:

  • Interpretation: Phosphorylated POLR2A may localize to specific nuclear regions

  • Validation: Co-stain with markers of transcription factories or specific genome regions

Resolution strategies:

• Sequential probing approach:

  • Strip and reprobe the same membrane with both antibodies

  • Always probe for phospho-forms first, then strip and reprobe for total protein

• Parallel sample analysis:

  • Run identical samples on separate blots for phospho and total detection

  • Ensure identical loading and transfer conditions

• Quantification method:

  • For Western blots, calculate the ratio of phospho-POLR2A to total POLR2A

  • For imaging, measure co-localization coefficients between phospho and total signals

When using both phospho-specific (such as NB100-1805) and total POLR2A antibodies, it's crucial to optimize stripping conditions to avoid epitope damage or incomplete stripping, which can lead to false interpretations .

What controls should I include when using POLR2A antibodies in different applications?

Proper controls are essential for ensuring valid and interpretable results when using POLR2A antibodies. Here are application-specific control recommendations:

Controls for Western blot:

• Positive controls:

  • Well-characterized cell lines with known POLR2A expression (HeLa, HepG2, MCF-7)

  • Recombinant POLR2A protein (full-length or domain-specific)

• Negative controls:

  • POLR2A siRNA/shRNA treated samples

  • For phospho-specific antibodies: Phosphatase-treated samples

• Specificity controls:

  • Peptide competition assays with phosphorylated and non-phosphorylated peptides

  • Secondary antibody-only controls to assess background

Controls for ChIP/ChIP-seq:

• Positive genomic regions:

  • Constitutively expressed genes (GAPDH, ACTB)

  • Genes known to be actively transcribed in your model system

• Negative genomic regions:

  • Silent genes (myoglobin in non-muscle cells)

  • Repressed chromatin (Sat2 satellite repeats)

• Antibody controls:

  • IgG (matched isotype) negative control

  • Input DNA (pre-immunoprecipitation) for normalization

Controls for IHC/IF:

• Tissue controls:

  • Tissues with known high (proliferating cells) and low (quiescent cells) transcriptional activity

  • Serial sections stained with total and phospho-specific antibodies

• Technical controls:

  • Antibody omission controls

  • Blocking peptide controls for phospho-specific antibodies

Experimental data from ChIP assays using the C.15200005 antibody demonstrated 30-40% recovery at active GAPDH and ACTB genes compared to <5% recovery at the inactive myoglobin gene and Sat2 satellite repeats, confirming antibody specificity .

How can I use POLR2A antibodies to study transcriptional pausing and elongation dynamics?

Transcriptional pausing and elongation dynamics are critical regulatory mechanisms in gene expression. POLR2A antibodies provide powerful tools for investigating these processes:

Methodological approach:

• Differential phosphorylation analysis:

  • Use pSer5 antibodies to detect initiated/paused polymerase (enriched at promoter-proximal regions)

  • Use pSer2 antibodies to detect elongating polymerase (enriched in gene bodies)

  • Calculate pSer2/pSer5 ratios to assess pausing release and elongation efficiency

• ChIP-seq with multiple antibodies:

  • Perform parallel ChIP-seq with pSer2, pSer5, and total POLR2A antibodies

  • Analyze the distribution patterns:

    • Promoter-proximal enrichment of pSer5 indicates pausing

    • Gene body enrichment of pSer2 indicates active elongation

    • Calculate traveling ratios (TR = promoter signal/gene body signal)

• Drug response studies:

  • Treat cells with transcription elongation inhibitors (e.g., DRB, flavopiridol)

  • Monitor changes in pSer2/pSer5 ratios and distribution patterns

  • Correlate with nascent RNA production (e.g., using GRO-seq or PRO-seq)

• Single-cell Western analysis:

  • Use the single-cell Western approach with POLR2A antibodies (100 μg/mL)

  • Analyze cell-to-cell variability in phosphorylation states

  • Correlate with cell cycle markers or differentiation status

This approach has been validated in multiple studies, with antibodies like NB100-1805 (pSer2) and C.15200005 (pSer2) providing reliable detection of elongating polymerase across diverse experimental systems .

What are the considerations for studying POLR2A in different species and model systems?

POLR2A is highly conserved across eukaryotes, making cross-species studies valuable but requiring specific considerations:

Species compatibility and validation:

Model system-specific approaches:

• Cell lines:

  • Human: HeLa, HepG2, MCF-7, A431 cells extensively validated

  • Mouse: NIH/3T3, HT-2 cell lines confirmed

  • Optimize extraction methods based on cell type (adherent vs. suspension)

• Tissue analysis:

  • For IHC: Optimize antigen retrieval methods (citrate buffer pH 6.0 or TE buffer pH 9.0)

  • Species-specific fixation times may require adjustment

  • Background levels vary across tissues; pancreatic tissues show defined nuclear localization

• Developmental studies:

  • Consider stage-specific expression and phosphorylation patterns

  • Mouse teratoma models validated for developmental expression patterns

When working with non-mammalian models, preliminary validation via Western blot is essential before proceeding to more complex applications like ChIP-seq. The high conservation of POLR2A means most mammalian-targeted antibodies will work across species, but the specific phosphorylation patterns may vary in timing and distribution .

How can POLR2A antibodies be used to investigate transcription-coupled processes like mRNA processing and chromatin remodeling?

POLR2A antibodies serve as powerful tools for studying the integration of transcription with other nuclear processes:

Integrated experimental approaches:

• Sequential ChIP (Re-ChIP) for co-occupancy analysis:

  • First IP: Use POLR2A antibodies (total or phospho-specific)

  • Second IP: Use antibodies against:

    • mRNA processing factors (splicing, capping, polyadenylation)

    • Chromatin modifiers (methyltransferases, acetylases)

    • Chromatin remodelers (SWI/SNF, ISWI complexes)

  • Analysis reveals co-occupancy and potential interaction

• Proximity Ligation Assay (PLA):

  • Detected as a cited application for POLR2A antibodies

  • Identifies proteins in close proximity (<40 nm) to POLR2A in situ

  • Combine phospho-specific POLR2A antibodies with antibodies against regulatory factors

  • Quantify interaction events using fluorescent foci counting

• IP-Mass Spectrometry:

  • Use POLR2A antibodies for immunoprecipitation (2-10 μg/mg protein)

  • Identify co-precipitating factors by mass spectrometry

  • Compare interactome differences between phosphorylation states

• ChIP-seq integration with other genomic data:

  • Overlay POLR2A ChIP-seq with:

    • RNA-seq for correlation with gene expression

    • ChIP-seq for histone modifications

    • ATAC-seq for chromatin accessibility

  • Identify coordination between transcription and chromatin states

These approaches have been validated using various POLR2A antibodies. For instance, proximity ligation assays using phospho-specific POLR2A antibodies have revealed spatial relationships between elongating polymerase and mRNA processing factors . Similarly, IP methods using 2-10 μg/mg of antibody successfully capture POLR2A and associated factors .

How are POLR2A antibodies being used in single-cell transcription studies?

The advent of single-cell technologies has opened new avenues for POLR2A antibody applications:

Single-cell methodological approaches:

• Single-cell Western blotting:

  • Validated approach using POLR2A antibodies at 100 μg/mL concentration

  • Enables detection of phosphorylation states in individual cells

  • Reveals cell-to-cell heterogeneity in transcriptional states

  • Correlate with cell cycle phase or differentiation status

• CUT&Tag and CUT&RUN adaptations:

  • Emerging applications of POLR2A antibodies for single-cell epigenomic profiling

  • Requires optimization of antibody concentrations for cellular permeability

  • Enables mapping of polymerase occupancy in rare cell populations

• Imaging-based approaches:

  • Immunofluorescence combined with RNA-FISH

  • Correlates polymerase phosphorylation state with nascent transcript production at single-gene resolution

  • Requires highly specific antibodies with low background (e.g., NB100-1805)

• Single-cell multi-omics integration:

  • Combining protein detection (using POLR2A antibodies) with scRNA-seq

  • CITE-seq adaptations for detecting POLR2A modifications alongside transcriptome

These emerging approaches extend beyond traditional bulk assays to reveal the heterogeneity in transcriptional states within seemingly homogeneous populations. The single-cell Western blot application, validated with antibodies like NB100-1805, represents a significant advancement in understanding transcriptional dynamics at single-cell resolution .

What are the considerations for using POLR2A antibodies in neuroscience and cancer research?

POLR2A antibodies have specific applications and considerations in specialized research fields:

Neuroscience applications:

• Activity-dependent transcription:

  • Monitor activity-induced phosphorylation changes in neurons

  • Track transcriptional dynamics during memory formation

  • Consider penetration issues in fixed brain tissues; optimize antigen retrieval

• Neuronal development:

  • Study transcriptional waves during neurogenesis and differentiation

  • Track changes in phosphorylation patterns during axon/dendrite development

  • Combine with neuronal markers for cell type-specific analysis

Cancer research applications:

• Transcriptional signatures in tumors:

  • Compare POLR2A phosphorylation patterns between tumor and normal tissues

  • Validated antibodies in cancer cell lines include AF6160 (HeLa), 20655-1-AP (human pancreatic cancer)

  • Consider tumor heterogeneity when interpreting bulk results

• Response to transcription-targeting therapeutics:

  • Monitor CDK inhibitor effects on Ser2/Ser5 phosphorylation

  • Track global transcriptional changes during treatment response

  • Combine with proliferation markers to distinguish direct vs. indirect effects

• Predictive/prognostic biomarker development:

  • Assess correlation between POLR2A phosphorylation patterns and patient outcomes

  • Optimize IHC protocols for clinical specimens

  • Validated in human pancreatic cancer tissue using 20655-1-AP antibody

When working with patient-derived samples, consider fixation effects on epitope accessibility. For neuroscience applications, background staining can be an issue; validated phospho-specific antibodies like NB100-1805 have shown good specificity in complex tissues like mouse teratoma .

How can computational approaches enhance the interpretation of POLR2A antibody data?

Computational analyses significantly enhance the value of POLR2A antibody data, particularly for genome-wide studies:

Advanced computational approaches:

• ChIP-seq data analysis pipelines:

  • Specialized peak calling for broad POLR2A signals vs. sharp TF binding sites

  • Calculate pausing indices: promoter-proximal signal vs. gene body signal

  • Metagene analyses to generate aggregate profiles across genes

  • Combine pSer2 and pSer5 data to identify paused vs. actively transcribing genes

• Integration with chromatin state data:

  • Correlate POLR2A occupancy with histone modification patterns

  • Define chromatin environments associated with different polymerase states

  • Machine learning approaches to predict POLR2A binding from chromatin features

• Network analysis of POLR2A-regulated genes:

  • Identify transcription factor networks associated with paused vs. active genes

  • Gene ontology enrichment analysis for biological process interpretation

  • Pathway analysis to identify cellular functions regulated by specific POLR2A states

• Visualization tools for multi-antibody datasets:

  • Genome browsers with custom tracks for different phospho-forms

  • Scatter plots comparing pSer2 vs. pSer5 enrichment to identify gene clusters

  • Heatmaps aligning genes by expression level, pausing index, or other features

These computational approaches transform antibody-generated data into biological insights. For example, ChIP-seq data generated using phospho-specific antibodies like C.15200005 can be analyzed to reveal genome-wide patterns of transcriptional regulation, identifying groups of genes with similar regulatory mechanisms .