POLR2G Antibody

What is POLR2G and what cellular functions does it perform?

POLR2G is a critical subunit of RNA polymerase II with a calculated molecular weight of 19 kDa (172 amino acids), though its observed molecular weight in experimental conditions typically ranges from 20-25 kDa . This subunit serves two primary functions: it participates in transcription initiation and helps stabilize transcribing polymerase molecules during the elongation phase . As part of the RNA polymerase II complex, POLR2G contributes to the synthesis of messenger RNA in eukaryotic cells, making it essential for gene expression regulation .

The protein is encoded by the POLR2G gene (GenBank Accession Number: BC112164, NCBI Gene ID: 5436) and has been well-conserved across species, indicating its fundamental importance in transcriptional processes . Understanding POLR2G's structure and function provides insights into the mechanics of gene regulation and expression, particularly in contexts where transcriptional pausing serves as a rate-limiting step.

Commercial POLR2G antibodies demonstrate reactivity across several species:

The high sequence conservation of POLR2G across species enables cross-reactivity of many antibodies. When working with species not directly validated, researchers should consider sequence homology as a predictor of potential reactivity . For critical experiments with non-validated species, preliminary validation through Western blot or other applicable techniques is advisable to confirm antibody performance.

What is the recommended storage and handling protocol for POLR2G antibodies?

For optimal stability and performance of POLR2G antibodies, following proper storage and handling protocols is crucial:

• Short-term storage: Store at 4°C

• Long-term storage: Store at -20°C, avoiding freeze/thaw cycles

• Most commercial POLR2G antibodies are provided in a liquid form with storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies are typically stable for one year after shipment when stored properly

• Aliquoting is generally unnecessary for -20°C storage in the manufacturer's buffer

• Some preparations (20μl sizes) may contain 0.1% BSA, while others are specifically BSA-free

It's important to note that sodium azide in the storage buffer is toxic and should be handled with appropriate precautions. Additionally, repeated freeze/thaw cycles should be avoided as they can lead to antibody degradation and reduced performance in experimental applications.

What molecular weight should be expected when detecting POLR2G in Western blot experiments?

When performing Western blot experiments with POLR2G antibodies, researchers should expect to observe bands at the following molecular weights:

• Calculated molecular weight: 19 kDa (172 amino acids)

• Observed molecular weight: 20-25 kDa range

The discrepancy between calculated and observed molecular weights is common in protein detection and may be attributed to several factors including post-translational modifications, relative charges, and other experimental factors that can affect protein migration in gels .

When interpreting Western blot results, researchers should consider that the observed molecular weight may vary based on the specific cell line or tissue being analyzed, the experimental conditions (including gel percentage, running buffer, and transfer method), and any post-translational modifications present in the target protein.

What are the optimal conditions for using POLR2G antibodies in chromatin immunoprecipitation (ChIP) experiments?

While the search results don't specifically address ChIP protocols for POLR2G antibodies, optimal conditions can be extrapolated from RNA polymerase II ChIP methodologies and general antibody principles:

For successful POLR2G ChIP experiments, researchers should consider:

When analyzing ChIP-seq data, researchers should correlate POLR2G binding patterns with transcriptional pausing indices, as POLR2G plays a role in promoter-proximal Pol II pausing, a key rate-limiting step for gene expression .

How can researchers effectively study POLR2G's role in promoter-proximal Pol II pausing?

POLR2G's involvement in transcription initiation and stabilization of polymerase molecules during elongation makes it relevant to studies of promoter-proximal pausing. To effectively investigate this role:

• Quantitative pausing analysis: Use Global-Run-On-sequencing (GRO-seq) to quantify transcriptional pausing at protein-coding genes with the pausing index (PI) or traveling ratio metric . Define the PI as the log-ratio of signals in the promoter-proximal region versus the gene body, using appropriate window sizes (e.g., sharp TSS window size of 3 bp ranging 1 bp up- and downstream of the TSS) .

• Integration with expression data: Correlate pausing indices with gene expression profiles from RNA-seq data. High PIs typically correlate with low gene expression (negative correlation, ρ = -0.68 in K562 cells and ρ = -0.66 in HepG2 cells) .

• Immunoprecipitation studies: Use POLR2G antibodies in immunoprecipitation experiments to isolate protein complexes and identify interaction partners involved in the pausing mechanism . This can reveal how POLR2G contributes to the stability of paused polymerase complexes.

• Machine learning approaches: Apply predictive models, as described in the literature, to analyze how POLR2G contributes to determining the extent of Pol II pausing in conjunction with other DNA and RNA-binding trans-acting factors .

• Cell line considerations: When designing experiments, note that different cell lines may exhibit distinct patterns of Pol II pausing. Studies have established methodologies for various cell lines including K562, HepG2, and HeLa .

By combining these approaches, researchers can elucidate POLR2G's specific contributions to transcriptional regulation through the mechanism of promoter-proximal pausing.

What strategies should be employed when using POLR2G antibodies for co-immunoprecipitation of associated transcription factors?

Co-immunoprecipitation (Co-IP) using POLR2G antibodies can reveal important protein-protein interactions within the transcriptional machinery. For optimal results, consider the following strategies:

When analyzing co-immunoprecipitated proteins, pay particular attention to other RNA Polymerase II subunits and known transcription elongation factors to understand POLR2G's specific role in transcriptional complexes.

How can researchers detect post-translational modifications of POLR2G using specific antibodies?

While the search results don't specifically mention post-translational modifications (PTMs) of POLR2G, researchers interested in studying these modifications should consider the following approaches:

• Modification-specific antibodies: Where available, use antibodies specifically raised against known or predicted PTMs of POLR2G. Common modifications to investigate include phosphorylation, acetylation, methylation, and ubiquitination.

• Two-dimensional Western blotting: First separate proteins by isoelectric point (first dimension) and then by molecular weight (second dimension). This can reveal charge variants of POLR2G resulting from PTMs.

• Phosphorylation detection:

  • Use phospho-specific antibodies if available

  • Employ phosphatase treatment of parallel samples to confirm phosphorylation

  • Use Phos-tag™ acrylamide gels to enhance separation of phosphorylated proteins

• Mass spectrometry validation: After immunoprecipitation with POLR2G antibodies, subject the purified protein to mass spectrometry analysis to identify and characterize PTMs.

• Enrichment strategies: Use phospho-enrichment techniques (TiO₂, IMAC) or other modification-specific enrichment approaches before Western blotting with general POLR2G antibodies.

• Cell treatment conditions: Compare POLR2G modification status under different cellular conditions, such as transcriptional activation, stress response, or cell cycle phases to identify condition-specific PTMs.

• Kinase/enzyme inhibitors: Use specific inhibitors to block particular modification pathways and observe the effect on POLR2G migration patterns in Western blots.

When interpreting results, remember that the observed molecular weight of POLR2G (20-25 kDa) differs from its calculated molecular weight (19 kDa), which could be partially explained by PTMs affecting protein migration .

What methodologies are recommended for studying POLR2G in the context of RNA polymerase II elongation dynamics?

To investigate POLR2G's role in RNA polymerase II elongation dynamics, researchers should consider these advanced methodological approaches:

• Nascent RNA sequencing techniques: Utilize Global-Run-On sequencing (GRO-seq) to capture nascent RNA fragments during transcription, enabling assessment of Pol II productivity and pausing dynamics . This allows calculation of pausing indices (PI) as the log-ratio between promoter-proximal signals and gene body signals.

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq): Compare POLR2G occupancy with other Pol II subunits to identify elongation-specific functions. Particular attention should be paid to the distribution of POLR2G at transition zones between initiation and productive elongation.

• Precision nuclear run-on sequencing (PRO-seq): This higher-resolution variant of GRO-seq can provide base-pair resolution of RNA polymerase positions, offering insights into the precise role of POLR2G during the transition from pausing to productive elongation.

• Single-molecule approaches: Techniques like single-molecule real-time imaging of transcription can reveal how POLR2G contributes to polymerase dynamics at the individual molecule level.

• CRISPR-mediated mutagenesis: Generate POLR2G mutants affecting specific domains to dissect their contributions to elongation stability. Combine with the above sequencing approaches to assess functional consequences.

• Integrated data analysis: Apply machine learning models that incorporate large-scale genome and transcriptome binding maps to predict how POLR2G interactions determine pausing and elongation behavior . Such models can accurately predict the extent of promoter-proximal Pol II pausing and reveal the relative importance of POLR2G compared to other factors.

• Inhibitor studies: Use transcription elongation inhibitors (e.g., DRB, flavopiridol) in combination with POLR2G antibody-based detection methods to isolate specific steps in the elongation process.

These approaches, when used in combination, can provide a comprehensive understanding of POLR2G's contributions to the complex process of transcriptional elongation and its regulation.