POLR2G Antibody, HRP conjugated

What is POLR2G and its role in transcription?

POLR2G (also known as RPB7) is the 19.294 kDa G subunit of RNA polymerase II, the enzyme responsible for synthesizing mRNA in eukaryotes. It belongs to the Eukaryotic RPB7/RPC8 RNA polymerase subunit family and plays a crucial role in the assembly and stability of the RNA polymerase II complex . This protein is encoded by the human POLR2G gene, with alternative names including RPB7, hRPB19, and hsRPB7 . POLR2G is primarily localized in the nucleus and participates in transcription factories, which are focal points of active transcription within the nucleus .

What applications are POLR2G antibodies suitable for?

POLR2G antibodies are validated for multiple applications including:

• Western blotting (WB) - for protein detection in cell/tissue lysates

• Enzyme-linked immunosorbent assay (ELISA) - for quantitative detection

• Flow cytometry (FCM) - for analyzing protein expression at the cellular level

• Immunofluorescence - for visualizing the subcellular localization

HRP-conjugated versions offer direct enzyme detection capability without requiring secondary antibodies, streamlining experimental workflows and potentially reducing background in detection systems.

What is the significance of the CTD phosphorylation in RNA polymerase II function?

The carboxyl-terminal domain (CTD) of RNA polymerase II contains multiple heptapeptide repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that undergo phosphorylation during different stages of transcription . Ser5 phosphorylation is associated with transcription initiation and is enriched at transcription start sites, while Ser2 phosphorylation is linked to elongation . This phosphorylation pattern regulates RNA polymerase II activity, mediating interactions with factors involved in mRNA processing and chromatin modification. Studies have shown that CTD phosphorylation can promote a switch from associating with mediator condensates to splicing factor condensates , highlighting its importance in coordinating transcription with co-transcriptional processes.

How can I distinguish between actively elongating and paused RNA polymerase II using POLR2G antibodies?

To differentiate between elongating and paused RNA polymerase II complexes:

• Use combinatorial antibody approaches targeting both POLR2G and phosphorylation-specific epitopes.

• Pair POLR2G antibody with antibodies against Ser2-phosphorylated CTD to identify elongating polymerase, as Ser2 phosphorylation is specifically associated with the elongation phase .

• Compare with Ser5 phosphorylation patterns, which are enriched at promoter regions and decrease as polymerase transcribes beyond the promoter .

• Consider chromatin immunoprecipitation (ChIP) assays to map POLR2G distribution along gene bodies, correlating with phosphorylation patterns to identify paused versus elongating complexes.

Research indicates that "unlike some mammalian studies, Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region" . This suggests careful interpretation is required when using phosphorylation status alone to determine polymerase activity state.

What controls should be included when performing ChIP experiments with POLR2G antibodies?

When designing ChIP experiments with POLR2G antibodies, incorporate these essential controls:

• Input control: Reserve a portion of chromatin before immunoprecipitation to normalize for differences in starting material

• Negative control regions: Include primers for genomic regions not expected to bind RNA polymerase II

• Positive control regions: Target housekeeping genes like GAPDH or PPIA that show consistent RNA polymerase II occupancy

• IgG control: Perform parallel immunoprecipitation with non-specific IgG from the same species as the POLR2G antibody

• Technical replicates: Perform at least three biological replicates to ensure reproducibility

• Antibody validation: Validate the specificity of the POLR2G antibody through Western blot analysis prior to ChIP experiments

For HRP-conjugated antibodies specifically, additional controls should address potential interference from the HRP moiety with chromatin binding.

How do phosphorylation patterns of RNA polymerase II differ between yeast and mammals?

Recent research suggests that changes in CTD phosphorylation patterns during transcription may be more conserved between yeast and humans than previously recognized . Key similarities and differences include:

What is the optimal protocol for Western blotting using HRP-conjugated POLR2G antibodies?

For optimal Western blot results with HRP-conjugated POLR2G antibodies:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • Centrifuge at 14,000×g for 15 minutes at 4°C to remove debris

• Gel electrophoresis and transfer:

  • Load 20-30 μg of total protein per lane

  • Separate proteins using 12% SDS-PAGE (optimal for the 19.294 kDa POLR2G protein)

  • Transfer to PVDF membrane at 100V for 1 hour

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated POLR2G antibody to 1:1000 in blocking buffer

  • Incubate membrane overnight at 4°C with gentle rocking

• Detection:

  • Wash membrane 3× with TBST, 5 minutes each

  • Develop using ECL substrate

  • Expose to X-ray film or capture images using a digital imager

Since HRP is directly conjugated to the primary antibody, no secondary antibody incubation is required, reducing protocol time and potentially decreasing background signal.

How can I troubleshoot non-specific binding when using POLR2G antibodies?

When encountering non-specific binding with POLR2G antibodies:

• Antibody dilution optimization:

  • Test a range of dilutions from 1:500 to 1:5000 to determine optimal signal-to-noise ratio

  • HRP-conjugated antibodies may require higher dilutions than unconjugated versions

• Blocking optimization:

  • Try alternative blocking agents (BSA, casein, commercial blockers)

  • Increase blocking time to 2 hours or overnight at 4°C

• Epitope considerations:

  • Verify antibody specificity for the C-terminal region (amino acids 125-154) of POLR2G

  • Consider testing antibodies targeting different epitopes if persistent cross-reactivity occurs

• Sample preparation:

  • Include phosphatase inhibitors if studying phosphorylated forms of RNA polymerase II

  • Pre-clear lysates with Protein A/G beads to remove non-specific binding proteins

• Validation techniques:

  • Confirm specificity using knockout/knockdown controls

  • Perform peptide competition assays using the immunizing peptide (DDEIRLKIVGTRVDKC sequence)

These methodological refinements should significantly reduce non-specific binding while maintaining sensitivity for the target protein.

What are the considerations for using POLR2G antibodies in live cell imaging studies?

When implementing POLR2G antibodies for live cell imaging:

• Cell delivery methods:

  • Standard antibodies including HRP-conjugated versions cannot penetrate intact cell membranes

  • Consider genetically encoded alternatives like the modification-specific intracellular antibody (mintbody) approach described by Uchino et al.

  • For conventional antibodies, microinjection or cell-penetrating peptide conjugation may be required

• Mintbody approach:

  • "To detect RNAP2 Ser2ph conveniently without protein loading, a genetically encoded live-cell probe can be developed, which consists of the single-chain variable fragment (scFv) of the specific antibody and superfolder GFP (sfGFP)"

  • This approach allows visualization of transcription "factories" in living cells

• Imaging considerations:

  • For standard fluorescence microscopy, resolution limitations may prevent visualization of individual POLR2G foci

  • "Recent confocal and 3D stimulated emission depletion microscopy has enabled detection of single elongating RNAP2 foci in living cells"

  • Consider photobleaching recovery experiments (FRAP) to study kinetics of different RNAP2 fractions

• Physiological relevance:

  • Monitor cells for stress responses that might alter transcription patterns

  • Maintain physiological temperature and pH during imaging

  • Consider the impact of mitosis on POLR2G distribution, as "most RNAP2 transcription is repressed during mitosis in mammalian cells"

Live cell imaging approaches provide unique insights into the dynamic behavior of RNA polymerase II that complement traditional biochemical analyses.

How should I interpret changes in POLR2G signal during different cell cycle phases?

When analyzing POLR2G distribution across the cell cycle:

• Interphase patterns:

  • Expect numerous nuclear foci representing transcription "factories"

  • The number and intensity of foci correlate with transcriptional activity

• Mitotic changes:

  • "In cells that started chromosome condensation at the onset of prophase, RNAP2 Ser2ph-mintbody foci were observed around the edge or outside the condensing chromosomes"

  • Most RNA polymerase II transcription is repressed during mitosis through release of the elongation complex

  • After cytokinesis, "RNAP2 Ser2ph-mintbody became concentrated in foci, and the number of foci gradually increased in early G1"

• Quantitative assessment:

  • Measure the number, intensity, and distribution of POLR2G foci throughout the cell cycle

  • Normalize to total nuclear area to account for changes in nuclear size

  • Track individual foci over time to determine persistence and mobility

• Correlation with gene activity:

  • Combine with nascent RNA labeling techniques to correlate POLR2G signal with active transcription

  • Consider gene-specific approaches for genes of interest

These patterns are consistent with the established model of transcriptional silencing during mitosis with gradual reactivation as cells enter G1 phase.

What can the co-localization of POLR2G with other factors reveal about transcriptional regulation?

Co-localization analysis of POLR2G with other nuclear factors provides insight into transcriptional mechanisms:

• Initiation factors:

  • Co-localization with Ser5-phosphorylated RNA polymerase II indicates transcription initiation sites

  • Low co-localization with Ser2-phosphorylated CTD suggests separation from elongation complexes

• Elongation factors:

  • "RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation"

  • Key elongation factors include CDK9, CDK12, and components of the Paf1 complex like LEO1

• RNA processing factors:

  • Co-localization with SRSF1 (serine/arginine-rich splicing factor 1) suggests coupling of transcription with RNA splicing

  • CTD phosphorylation promotes "a condensate preference switch from the mediator to splicing factor condensates"

• Enhancer elements:

  • Association with BRD4 and p300 histone acetyltransferase indicates interaction with enhancer elements

  • These interactions help identify genes under active regulation

Quantitative co-localization analysis should include Pearson's correlation coefficient and Manders' overlap coefficient calculations to determine the strength and extent of association between POLR2G and other factors.

How can I integrate POLR2G ChIP-seq data with other genomic datasets?

For comprehensive integration of POLR2G ChIP-seq with other genomic data:

• Data preprocessing steps:

  • Perform standard quality control and normalization

  • Use appropriate peak calling algorithms (MACS2 recommended)

  • Generate normalized coverage tracks for visualization

• Multi-omic integration approaches:

  • Compare POLR2G occupancy with POLR2A/B to assess complete polymerase complex formation

  • Overlap with CTD phosphorylation states (Ser2P, Ser5P) to determine elongation vs. initiation

  • Correlate with histone modifications (H3K4me3 at promoters, H3K36me3 in gene bodies)

  • Integrate with nascent RNA sequencing (GRO-seq, PRO-seq) to correlate occupancy with transcriptional output

• Analysis tools and visualizations:

  • Use deepTools for correlation heatmaps and profile plots

  • Apply multivariate analysis techniques (PCA, t-SNE) to identify patterns across datasets

  • Create genome browser tracks showing POLR2G occupancy alongside other features

• Biological interpretation:

  • Identify genes with promoter-proximal pausing (high promoter/gene body ratio)

  • Analyze changes in POLR2G distribution in response to stimuli or inhibitors

  • Compare patterns across different cell types or conditions

This integrated approach provides a comprehensive view of transcriptional regulation beyond what any single dataset can reveal.

What are the differences between polyclonal and monoclonal POLR2G antibodies?

Understanding the differences between polyclonal and monoclonal POLR2G antibodies is crucial for experimental design:

The polyclonal anti-POLR2G generated from rabbits immunized with "a KLH conjugated synthetic peptide between 125-154 amino acids from the C-terminal region of human POLR2G" offers broad epitope recognition, while monoclonal versions provide more consistent results across experiments.

How do different conjugation methods affect POLR2G antibody performance?

The conjugation chemistry and approach significantly impact antibody performance:

• HRP conjugation considerations:

  • HRP conjugation typically uses periodate or maleimide chemistry

  • Optimal conjugation preserves antibody binding while maintaining enzymatic activity

  • HRP-conjugated antibodies show excellent signal amplification in Western blot and ELISA applications

• Impact on antibody properties:

  • Molecular size increases (HRP adds approximately 44 kDa)

  • Steric hindrance may affect epitope accessibility in certain applications

  • May alter optimal working dilutions compared to unconjugated antibodies

• Alternative conjugation options:

  • Fluorescent conjugates (FITC, Alexa Fluor) offer direct visualization

  • Biotin conjugation provides versatility with multiple detection systems

  • Enzyme alternatives like alkaline phosphatase offer different detection sensitivities

• Application-specific considerations:

  • For Western blotting: HRP conjugates provide excellent sensitivity with chemiluminescent substrates

  • For microscopy: Direct fluorophore conjugates eliminate secondary antibody steps

  • For multiplex applications: Consider using conjugates with minimal spectral overlap

Each conjugation method requires validation to ensure retention of specificity and sensitivity for the target epitope.

What are the storage and handling recommendations for maintaining POLR2G antibody activity?

To preserve POLR2G antibody functionality:

• Storage temperature recommendations:

  • "Maintain refrigerated at 2-8°C for up to 6 months"

  • "For long term storage store at –20°C in small aliquots to prevent freeze-thaw cycles"

  • Avoid storing at room temperature for extended periods

• Buffer considerations:

  • Optimal buffer: "PBS with 0.09% (W/V) sodium azide, pH7.4"

  • Sodium azide preserves antibody stability but is incompatible with HRP activity (relevant for working dilutions)

  • Maintain recommended antibody concentration (typically 0.5mg/ml)

• Handling practices:

  • Minimize freeze-thaw cycles by preparing single-use aliquots

  • Briefly centrifuge vials after thawing to collect liquid at the bottom

  • Use sterile technique when handling to prevent contamination

  • Allow antibody to reach room temperature before opening vials

• Working solution preparation:

  • Dilute only the amount needed for immediate use

  • Prepare working dilutions in buffer without sodium azide when using HRP-conjugated antibodies

  • Return the stock solution to proper storage conditions immediately after use

Following these guidelines will maximize antibody shelf-life and ensure consistent experimental results.

How are POLR2G antibodies being used to study transcription dynamics in living cells?

Recent advances in live-cell imaging with POLR2G and RNA polymerase II:

• Genetically encoded probes:

  • The development of "genetically encoded modification-specific intracellular antibody (mintbody) probe" allows visualization of RNAP2 phosphorylation in living cells

  • This approach enables tracking of "numerous foci, possibly representing transcription 'factories'"

• High-resolution dynamics:

  • "High-resolution single-molecule analyses using photoconvertible or photoactivatable RNAP2 have indicated the transient clustering of RNAP2 during initiation"

  • These approaches reveal interactions "in association with mediator condensates"

• Spatio-temporal organization:

  • Recent studies have enabled "visualization of the spatiotemporal organization of RNAP2 phosphorylation and mRNA synthesis"

  • This includes detailed kinetic analysis of RNAP2 Ser5 phosphorylation on single-copy genes

• Cell cycle progression:

  • Live imaging reveals that "RNAP2 Ser2ph-mintbody foci were observed around the edge or outside the condensing chromosomes" during prophase

  • Following division, foci concentration gradual increases as cells enter G1

These approaches have revolutionized our understanding of transcription as a dynamic process rather than a static event, revealing transient interactions and condensate formations that regulate gene expression.

What is the current understanding of the relationship between POLR2G and disease mechanisms?

While the search results don't directly address POLR2G in disease, the role of RNA polymerase II in transcriptional regulation has significant disease implications:

• Cancer biology:

  • Dysregulation of transcription machinery is a hallmark of many cancers

  • Changes in RNA polymerase II phosphorylation patterns affect gene expression programs

  • Understanding POLR2G function may provide insights into transcriptional addiction in cancer cells

• Neurodegenerative disorders:

  • RNA polymerase II function is critical for neuronal gene expression

  • Altered transcription dynamics have been implicated in conditions like Huntington's and Alzheimer's diseases

  • POLR2G antibodies can help elucidate these mechanisms

• Viral pathogenesis:

  • Many viruses interact with and manipulate the host transcription machinery

  • POLR2G detection can reveal how viral factors affect RNA polymerase II function

  • This understanding may guide development of antiviral strategies

• Developmental disorders:

  • Proper transcriptional regulation is essential for embryonic development

  • Mutations affecting RNA polymerase II components can lead to developmental abnormalities

  • POLR2G antibodies facilitate studies of transcriptional dynamics during development

Future research targeting POLR2G specifically may reveal its unique contributions to health and disease beyond the general functions of RNA polymerase II.

How can POLR2G antibodies be used in single-cell transcriptional profiling?

Integrating POLR2G detection with single-cell technologies:

• Single-cell immunofluorescence approaches:

  • POLR2G antibodies can identify active transcription sites within individual cells

  • Correlation with nascent RNA detection reveals cell-to-cell variability in transcription

  • Co-detection with cell type-specific markers identifies specialized transcriptional programs

• Integration with single-cell genomics:

  • Combining POLR2G ChIP with single-cell techniques reveals cell-specific occupancy patterns

  • CUT&Tag approaches with POLR2G antibodies can map polymerase occupancy in rare cell populations

  • Correlation with scRNA-seq data connects polymerase activity to transcriptional output

• Methodological adaptations:

  • Miniaturization of ChIP protocols for limited cell numbers

  • Use of highly sensitive detection methods for HRP-conjugated antibodies

  • Implementation of multiplexed antibody approaches to simultaneously detect multiple factors

• Analytical considerations:

  • Computational methods to deconvolute signals from cell mixtures

  • Trajectory analysis to map transcriptional dynamics during cell state transitions

  • Network analysis to identify coordinated changes in transcriptional programs

These approaches extend beyond bulk analysis to reveal the heterogeneity in transcriptional regulation at the single-cell level.

What novel research applications are emerging for POLR2G antibodies in transcription-related condensate studies?

POLR2G antibodies are providing insights into phase separation and biomolecular condensates:

• Transcriptional condensates:

  • "CTD phosphorylation can promote a condensate preference switch from the mediator to splicing factor condensates"

  • POLR2G antibodies help track RNA polymerase II association with these dynamic structures

• High-resolution microscopy applications:

  • "High-resolution single-molecule analyses using photoconvertible or photoactivatable RNAP2 have indicated the transient clustering of RNAP2 during initiation in association with mediator condensates"

  • Super-resolution approaches reveal nanoscale organization of transcription factories

• Multi-factor tracking:

  • Combined detection of POLR2G with mediator components, transcription factors, and RNA processing factors

  • Analysis of factor exchange rates within condensates using FRAP and related techniques

  • Correlation of condensate properties with transcriptional output

• Technological innovations:

  • CRISPR-mediated tagging of endogenous POLR2G for live imaging

  • Optogenetic approaches to manipulate condensate formation and stability

  • Computation modeling of factor interactions within transcriptional condensates

This emerging field is revealing how the physical organization of transcription machinery through phase separation contributes to gene regulation.

How do chromatin immunoprecipitation approaches differ when using POLR2G antibodies versus phosphorylation-specific antibodies?

Comparing ChIP strategies for RNA polymerase II components:

• Target specificity considerations:

  • POLR2G antibodies detect the total RNA polymerase II population regardless of CTD phosphorylation state

  • Phosphorylation-specific antibodies (Ser2P, Ser5P) identify subpopulations at different transcriptional stages

  • Combined approaches provide comprehensive view of polymerase distribution and activity

• Technical differences:

  • "An improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency" may be beneficial for both approaches

  • Crosslinking conditions may need optimization depending on target epitope accessibility

  • HRP-conjugated antibodies require modified elution protocols to prevent enzyme interference

• Data interpretation:

  • POLR2G ChIP reveals total polymerase occupancy along genes

  • Ser5P enrichment indicates promoter regions and initiation

  • Ser2P identifies elongating polymerase within gene bodies

  • Ratios between these signals can identify promoter-proximal pausing

• Comparative findings:

  • "Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat"

  • "Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region"

These complementary approaches provide a complete picture of RNA polymerase II dynamics during the transcription cycle.

What are the advantages and limitations of using antibody-based detection versus genetic tagging of POLR2G?

Comparing antibody-based and genetic tagging approaches:

The mintbody approach represents a hybrid solution that "consists of the single-chain variable fragment (scFv) of the specific antibody and superfolder GFP (sfGFP)" allowing detection of specific modifications in living cells .