POLR2G Antibody, FITC conjugated

What is POLR2G and why is it important in transcription research?

POLR2G (also known as RPB7) is a subunit of RNA polymerase II (Pol II), the multi-subunit enzyme responsible for the transcription of protein-coding genes. This 19 kDa subunit (sometimes referred to as RPB19) plays a crucial role in the structure and function of the RNA Pol II complex . Studying POLR2G is important for understanding fundamental transcriptional mechanisms, as it contributes to the assembly and stability of the RNA polymerase II complex. Recent research has demonstrated its presence in transcriptionally active biomolecular condensates (BMCs), suggesting its role in the spatial organization of transcription machinery within the nucleus .

What species reactivity can be expected with commercially available POLR2G antibodies?

Most commercially available POLR2G antibodies demonstrate reactivity with human POLR2G, while some also cross-react with mouse and rat orthologs . For instance, the mouse monoclonal antibody POLR2G (C-2) has been validated for detection of POLR2G from human, mouse, and rat origins . When selecting an antibody for your research, it is important to verify the specific species reactivity in the product documentation, as this can vary between different antibody clones and manufacturers. Cross-species reactivity should be experimentally validated if working with non-human models not explicitly listed in the antibody specifications.

How does the FITC conjugation benefit visualization of POLR2G in cellular contexts?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the POLR2G antibody, eliminating the need for secondary antibody detection steps in immunofluorescence applications. This conjugation enables visualization of POLR2G localization within cellular structures using fluorescence microscopy with excitation at approximately 495 nm and emission at around 519 nm. The direct conjugation is particularly advantageous for multi-color immunofluorescence experiments, co-localization studies with other nuclear factors, and when working with limiting tissue samples . FITC-conjugated POLR2G antibodies have been successfully used to visualize the co-localization of RNA Pol II machinery with DNA in transcriptionally active nuclear bodies .

What are the optimal storage conditions for maintaining FITC-conjugated POLR2G antibody activity?

To maintain optimal activity of FITC-conjugated POLR2G antibodies, store at -20°C to -80°C in the dark to prevent photobleaching of the FITC fluorophore . The antibody is typically supplied in a buffer containing preservatives (such as 0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) . Avoid repeated freeze-thaw cycles, as these can degrade both the antibody protein and the conjugated fluorophore. If frequent use is anticipated, consider aliquoting the antibody into single-use volumes upon receipt. For short-term storage (1-2 weeks), 4°C is acceptable, but protect from light. Always centrifuge the product briefly before opening the vial to ensure all material is at the bottom of the tube.

What applications are validated for POLR2G antibodies, and what starting dilutions are recommended?

POLR2G antibodies have been validated for multiple applications including:

The optimal dilution should be determined empirically for each specific application and experimental system . For novel applications, a titration experiment is strongly recommended.

How can POLR2G antibodies be incorporated into flow cytometry protocols for transcription studies?

For flow cytometry analysis of POLR2G chromatin binding, follow these methodological steps:

• Fix cells with 4% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with 0.25% Triton X-100 in PBS for 10 minutes

• Block with 5% BSA in PBS for 30 minutes

• Incubate with FITC-conjugated POLR2G antibody (typically 1:50-1:200 dilution) for 1 hour at room temperature or overnight at 4°C

• Wash three times with PBS

• For cell cycle analysis, counterstain with propidium iodide or DAPI

• Analyze using a flow cytometer with appropriate laser and filter settings for FITC detection

This method provides quantitative measurement of POLR2G levels across thousands of cells and can be combined with cell cycle analysis without requiring cell synchronization . For studying transcription dynamics, this approach can be combined with treatments using transcriptional inhibitors like DRB (5,6-Dichloro-1-beta-Ribo-furanosyl Benzimidazole) or proteasome inhibitors such as MG132 .

How can POLR2G antibodies be utilized to study biomolecular condensates in transcription?

To investigate POLR2G in the context of transcriptionally active biomolecular condensates (BMCs), implement the following experimental approach:

• Prepare nuclear extracts from your cell type of interest

• Mix the extracts with promoter DNA (e.g., CMV promoter) labeled with a fluorescent dye like Alexa Fluor 488

• Incubate the mixture to allow formation of preinitiation complexes

• Perform immunofluorescence using FITC-conjugated POLR2G antibody or other RNA Pol II subunit antibodies

• Visualize the condensates using confocal microscopy to assess co-localization of DNA and POLR2G

• For functional studies, perform parallel transcription assays using radioactive nucleotides to correlate condensate formation with transcriptional activity

This approach allows for direct visualization of POLR2G within transcriptionally active nuclear bodies and provides insights into the spatial organization of the transcription machinery . Control experiments should include promoter-less DNA constructs to demonstrate promoter dependency of condensate formation.

What controls should be included when using POLR2G antibodies in chromatin immunoprecipitation experiments?

When designing chromatin immunoprecipitation (ChIP) experiments with POLR2G antibodies, include these essential controls:

• Input DNA control: Set aside a portion (5-10%) of the chromatin before immunoprecipitation to normalize for differences in starting material

• Isotype control: Use an isotype-matched non-specific antibody (e.g., normal rabbit IgG for rabbit polyclonal POLR2G antibodies) to assess non-specific binding

• Positive control regions: Include primer sets for known RNA Pol II-bound regions (e.g., actively transcribed housekeeping genes)

• Negative control regions: Include primer sets for regions not expected to bind RNA Pol II (e.g., gene deserts or silenced genes)

• Technical replicates: Perform at least three technical replicates for each qPCR reaction

• Biological replicates: Use biological replicates to account for sample-to-sample variation

For POLR2G-specific validation, include a comparison with ChIP using antibodies against other RNA Pol II subunits to confirm co-occupancy at transcriptionally active sites. This comprehensive control strategy ensures reliable and interpretable ChIP results when studying POLR2G binding patterns across the genome.

How can POLR2G antibodies be used in combination with transcriptional inhibitors to study RNA Pol II dynamics?

To study RNA Pol II dynamics using POLR2G antibodies in combination with transcriptional inhibitors:

• Treat cells with specific inhibitors that target different phases of transcription:

  • DRB (5,6-Dichloro-1-beta-Ribo-furanosyl Benzimidazole) to arrest RNAPII in promoter proximal regions

  • α-amanitin for selective inhibition of RNA Pol II

  • Triptolide to inhibit TFIIH and prevent transcription initiation

• In parallel, treat cells with proteasome inhibitors (e.g., MG132) to prevent degradation of stalled polymerase

• Perform immunofluorescence or flow cytometry using FITC-conjugated POLR2G antibodies to visualize and quantify the changes in POLR2G localization and abundance

• For time-course experiments, harvest cells at various intervals following inhibitor treatment

• Complement with Western blotting to assess total POLR2G protein levels and chromatin fractionation to distinguish between soluble and chromatin-bound fractions

This methodological approach has revealed that promoter-proximal RNAPII undergoes degradation under normal conditions, which is enhanced following UV treatment . The combination of flow cytometry with POLR2G antibodies provides a powerful tool for studying the dynamics of RNA Pol II during transcription and in response to various cellular stresses.

What factors might affect the accuracy of POLR2G detection in immunofluorescence experiments?

Several factors can influence the accuracy of POLR2G detection in immunofluorescence experiments:

• Fixation method: Overfixation can mask epitopes while underfixation can result in poor structural preservation. For POLR2G, 4% paraformaldehyde fixation for 10-15 minutes is typically optimal.

• Antibody specificity: Validate antibody specificity using POLR2G knockdown or knockout cells as negative controls.

• FITC photobleaching: FITC is susceptible to photobleaching. Use anti-fade mounting media, minimize exposure to light during processing, and implement strategies to reduce illumination intensity and duration during imaging.

• Autofluorescence: Cellular components may exhibit autofluorescence in the same range as FITC. Include unstained controls and consider autofluorescence quenching reagents if necessary.

• Cross-reactivity: The antibody may cross-react with other RNA Pol II subunits. Verify specificity using peptide competition assays or alternative antibodies targeting different epitopes.

• Nuclear accessibility: Insufficient permeabilization can prevent antibody access to nuclear POLR2G. Optimize permeabilization conditions using detergents like Triton X-100.

• Protein extraction during processing: Harsh washing steps may extract nuclear proteins. Use gentle washing and consider performing parallel Western blots to verify retention of POLR2G during the immunofluorescence protocol.

Addressing these factors through careful protocol optimization will enhance the reliability of POLR2G detection in immunofluorescence applications.

How should researchers address potential discrepancies between POLR2G detection methods?

When faced with discrepancies between different POLR2G detection methods (e.g., Western blot shows presence but immunofluorescence shows weak signal), implement this systematic troubleshooting approach:

• Verify antibody functionality: Test the same antibody lot across multiple applications to ensure it performs as expected.

• Epitope accessibility: Different fixation and permeabilization conditions can affect epitope accessibility. Try alternative fixatives (e.g., methanol vs. paraformaldehyde) or permeabilization reagents.

• Protein conformation: The POLR2G epitope may be masked in certain assays due to protein folding or complex formation. Consider using antibodies targeting different regions of POLR2G.

• Expression levels: POLR2G might be expressed at levels below the detection threshold for certain methods. Consider signal amplification techniques for less sensitive methods.

• Cross-validation: Use orthogonal approaches such as RNA-seq to confirm POLR2G expression or mass spectrometry to verify protein presence.

• Subcellular fractionation: Perform nuclear/cytoplasmic fractionation to determine if POLR2G is primarily in a specific compartment that might be challenging to visualize with certain methods.

• Technical validation: Ensure all reagents are functional by including positive controls with known POLR2G expression patterns.

By systematically addressing these potential sources of discrepancy, researchers can reconcile apparently contradictory results and gain a more accurate understanding of POLR2G biology in their experimental system.

What patterns of POLR2G staining might indicate experimental artifacts versus biological phenomena?

Distinguishing between artifacts and genuine biological phenomena when analyzing POLR2G staining patterns requires careful consideration of the following characteristics:

If POLR2G appears in unexpected subcellular locations, verify by comparing multiple antibodies targeting different epitopes and correlate with functional transcription assays. True biological phenomena should be reproducible across different experimental conditions and detection methods .

How is POLR2G involved in the formation of transcriptionally active biomolecular condensates?

POLR2G, as a component of the RNA Polymerase II complex, has been implicated in the formation of transcriptionally active biomolecular condensates (BMCs) in vitro. These condensates form through phase transitions between promoter DNA and nuclear extract proteins, creating dense bodies approximately 0.5 to 1 μm in diameter with a macromolecular density of approximately 100 mg/ml . Experimental evidence shows that:

• POLR2G and other RNA Pol II components colocalize with promoter DNA (such as CMV promoter) within these bodies, as visualized using immunofluorescence techniques

• The formation of these condensates is promoter-dependent, though there appears to be a general DNA component as well

• The condensates are physically associated with transcription, as disruption of the condensates compromises transcriptional activity and vice versa

• These BMCs may represent a more physiologically relevant manifestation of the preinitiation complex/elongation machinery than traditional biochemical assays

This research suggests that POLR2G participates in creating a concentrated environment for transcription that mimics the dense nuclear environment in vivo. These findings open new avenues for studying transcriptional regulation in a context that better reflects physiological conditions .

What has research revealed about POLR2G degradation in response to cellular stress?

Recent research utilizing flow cytometry methods for analyzing RNA Polymerase II chromatin binding has revealed important insights about POLR2G regulation under stress conditions:

• Promoter proximal RNAPII (which includes the POLR2G subunit) undergoes degradation even under unperturbed, normal cellular conditions

• This degradation is significantly enhanced following UV treatment, suggesting a stress-responsive mechanism

• Using the transcriptional inhibitor DRB (5,6-Dichloro-1-beta-Ribo-furanosyl Benzimidazole), which arrests RNAPII in promoter proximal regions, in combination with the proteasome inhibitor MG132, researchers demonstrated that this degradation is mediated by the proteasome

• This degradation mechanism may represent a quality control process to clear stalled polymerase complexes from promoter regions

• The phenomenon has been observed through both flow cytometry and independent live-cell imaging approaches, strengthening the validity of these findings

These discoveries highlight the dynamic nature of POLR2G/RNA Pol II regulation and suggest potential roles in transcriptional stress responses and quality control mechanisms . Understanding these degradation pathways may provide insights into diseases associated with transcriptional dysregulation.

What emerging techniques are enhancing our ability to study POLR2G function in transcription dynamics?

Several cutting-edge techniques are advancing our understanding of POLR2G function in transcription dynamics:

• Flow cytometry-based chromatin binding analysis: This technique enables quantitative assessment of POLR2G binding across thousands of cells without requiring cell synchronization, allowing for cell cycle-specific analysis of transcription dynamics .

• In vitro reconstitution of biomolecular condensates: Researchers can now form transcriptionally active condensates containing POLR2G and other RNA Pol II components using only nuclear extracts and promoter DNA, providing a system to study phase separation in transcription regulation .

• Live-cell imaging with fluorescently tagged POLR2G: This approach allows real-time visualization of POLR2G dynamics during transcription, revealing degradation of promoter-proximal RNAPII after UV treatment.

• Cryo-electron microscopy: High-resolution structural studies are elucidating the precise positioning and interactions of POLR2G within the RNA Pol II complex during different stages of transcription.

• Proximity labeling techniques: BioID or APEX2 fused to POLR2G is enabling identification of transient interaction partners in living cells, providing new insights into the protein interaction network around POLR2G.

• Single-molecule tracking: This technique is revealing the diffusion dynamics and residence times of POLR2G at active genes, enhancing our understanding of transcription kinetics.

• Multi-omics integration: Combining POLR2G ChIP-seq with nascent RNA sequencing and proteomics is creating comprehensive views of how POLR2G contributes to transcriptional output.