RPB5 Antibody

What is RPB5 and what is its role in transcriptional regulation?

RPB5 (also known as POLR2E) is a common subunit of RNA polymerases I, II, and III that catalyzes the transcription of DNA into RNA using ribonucleoside triphosphates as substrates. According to structural studies, RPB5 forms part of the lower jaw of RNA polymerase II, with its N-terminal two-thirds exposed and positioned close to DNA downstream of the initiation site. This strategic positioning allows RPB5 to serve as a communicating surface that interacts with various transcriptional regulators, including TFIIF subunit RAP30, TFIIB, and viral proteins like Hepatitis B virus X protein (HBx) . The exposed domain of RPB5 plays a crucial role in facilitating these protein-protein interactions that modulate transcriptional activity, making it an important factor in both basal and activated transcription mechanisms.

Research has shown that RPB5 is essential for proper assembly and function of all three RNA polymerases, highlighting its fundamental importance in eukaryotic gene expression. This common subunit represents a potential regulatory node where various signaling pathways can converge to influence transcription across multiple polymerase systems .

What applications are RPB5 antibodies commonly used for?

RPB5 antibodies are versatile tools employed across multiple experimental applications in molecular and cellular biology research. Based on commercial product information and published studies, these antibodies are validated for numerous techniques:

For optimal IHC results, antigen retrieval with TE buffer at pH 9.0 is recommended, although citrate buffer at pH 6.0 may serve as an effective alternative . When establishing a new application, it is advisable to perform antibody titration to determine optimal concentration for your specific experimental system.

What species reactivity do commercial RPB5 antibodies demonstrate?

Commercial RPB5 antibodies exhibit reactivity across multiple species, reflecting the highly conserved nature of this essential RNA polymerase subunit. The reactivity profile varies between antibody products:

When selecting an antibody for cross-species applications, researchers should carefully evaluate the validation data for their species of interest. The high degree of conservation in RPB5 structure across species (noted between yeast and human homologs) suggests potential cross-reactivity even when not explicitly tested , but experimental validation is always recommended before proceeding with large-scale studies.

What is the molecular weight of RPB5 protein that should be detected?

When analyzing RPB5-interacting proteins, researchers should be aware of additional bands that may appear in immunoblots. For instance, RMP (RPB5-Mediating Protein, also known as URI), which directly interacts with RPB5, has an observed molecular weight of approximately 79 kDa and may co-immunoprecipitate with RPB5.

For validation purposes, positive Western blot detection of RPB5 has been confirmed in multiple cell lines including A549, HeLa, MCF-7, HepG2, and Jurkat cells, as well as in rat and mouse liver and spleen tissues . These validated samples serve as excellent positive controls when establishing RPB5 antibody performance in new experimental systems.

How should RPB5 antibodies be stored and handled for optimal performance?

• Storage temperature: Store at -20°C where antibodies remain stable for approximately one year after shipment .

• Storage buffer: Most RPB5 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: Generally unnecessary for -20°C storage, though aliquoting may be beneficial for frequently used antibodies to avoid repeated freeze-thaw cycles .

• Special considerations: Some smaller volume preparations (20μl sizes) may contain 0.1% BSA as a stabilizer .

• Working practice: Allow antibodies to equilibrate to room temperature before opening to prevent condensation that could dilute or contaminate the antibody.

• Dilution storage: Once diluted for working solutions, store at 4°C and use within 1-2 weeks for optimal performance.

For applications requiring conjugated antibodies, additional precautions may be necessary to protect fluorophores or enzymes from light exposure or oxidation. Always consult the specific product documentation for any unique storage or handling requirements.

How can RPB5 antibodies be used to investigate the relationship between RPB5 and RMP/URI?

RMP (RPB5-Mediating Protein), also known as URI (Unconventional prefoldin RBP5 Interactor), is a novel cellular protein that specifically interacts with RPB5 and functions as a transcriptional corepressor. This interaction occurs through the RPB5-binding region of RMP (amino acids 151-231) and the central part of RPB5, which overlaps with the HBx-binding region . RMP negatively modulates RNA polymerase II function and antagonizes the coactivator function of HBx by competitive binding to RPB5 .

To investigate this important regulatory interaction, researchers can employ several antibody-based approaches:

• Co-immunoprecipitation (Co-IP): Using anti-RPB5 antibodies for immunoprecipitation followed by Western blotting with anti-RMP antibodies can confirm and quantify this interaction under various experimental conditions. HeLa cells have been validated for positive IP detection of RPB5 .

• Chromatin Immunoprecipitation (ChIP): RPB5 antibodies can be used to identify genomic regions where RNA polymerase II containing RPB5 is bound. Comparing these profiles with RMP ChIP data can reveal regions where both proteins co-localize, suggesting functional interaction on chromatin.

• Sequential ChIP (ChIP-reChIP): This modified technique using both RPB5 and RMP antibodies sequentially can determine if both proteins simultaneously occupy the same genomic regions.

• Proximity Ligation Assay (PLA): Using RPB5 and RMP antibodies, this technique can visualize and quantify protein-protein interactions in situ, providing spatial information about where in the cell these proteins interact.

Research has shown that RMP is composed of 508 amino acid residues and contains an α-class prefoldin domain, the RPB5 interaction region, a long acidic sequence, and a conserved C-terminal sequence . RMP occurs as part of a complex with other prefoldin family members and RPB5, suggesting a role in coordinating transcriptional regulation with other cellular processes .

What are the critical mutations in RPB5 that affect its function, and how do they impact antibody recognition?

Mutational analysis of human RPB5 has identified specific residues critical for its interactions with regulatory proteins. Through systematic alanine scanning, researchers have mapped key regions essential for binding partners like HBx (Hepatitis B virus X protein):

• Critical sequence clusters: Four sequences (cm70, cm98, cm105, and cm112) within the central part of RPB5 (amino acids 53-136) are critical for HBx binding both in vivo and in vitro .

• Essential single residues: Point mutations at V74A, I104A, T111A, and S113A completely abolished the interaction of HBx with RPB5 .

These mutations potentially impact antibody recognition depending on the epitope targeted by the antibody. For antibodies whose epitopes overlap with these mutation sites, binding efficiency may be significantly reduced or eliminated.

When using RPB5 antibodies to study mutant variants, researchers should consider:

• Epitope mapping: Determine whether your antibody's epitope overlaps with known functional mutation sites

• Multiple antibody approach: Use antibodies targeting different RPB5 epitopes to ensure detection

• Recombinant tagging: Consider using tagged RPB5 variants and tag-specific antibodies as an alternative detection strategy

• Validation controls: Include wild-type RPB5 as a positive control in experiments with mutants

Understanding these structure-function relationships is critical for interpreting antibody-based experiments involving RPB5 mutants and their impact on transcriptional regulation.

How can RPB5 antibodies be utilized to study the assembly of RNA polymerase complexes?

RPB5 antibodies provide valuable tools for investigating the assembly and composition of RNA polymerase complexes. Research has shown that the prefoldin protein Bud27 (yeast homolog of human URI/RMP) mediates the assembly of all three eukaryotic RNA polymerases (I, II, and III) through its interaction with RPB5 .

Several experimental approaches utilizing RPB5 antibodies can illuminate RNA polymerase assembly:

• Co-immunoprecipitation coupled with mass spectrometry: Immunoprecipitating RPB5 and analyzing associated proteins can reveal differences in complex composition under various experimental conditions. This approach has shown that increasing the dosage of RPB5 corrects RNA polymerase assembly defects caused by the absence of Bud27 in yeast .

• Differential nuclear localization studies: Immunofluorescence using RPB5 antibodies can track the subcellular localization of RNA polymerase complexes. Research demonstrates that Bud27 deletion affects RNA polymerase nuclear localization, and both RPB5 and BUD27 overexpression can rescue this defect .

• Density gradient centrifugation with immunoblotting: This technique can separate RNA polymerase complexes at different assembly stages, with RPB5 antibodies used to track RPB5 incorporation into these complexes.

• Native gel electrophoresis: Combined with RPB5 immunoblotting, this approach can analyze intact RNA polymerase complexes and subcomplexes.

Research findings reveal that RPB5 plays a central role in polymerase assembly, as demonstrated by the fact that RPB5 overexpression corrects temperature sensitivity, rapamycin sensitivity, nuclear RNA polymerase localization, and RNA polymerase assembly in cells lacking Bud27 . This highlights the potential of RPB5 antibodies as critical tools for studying the dynamics and regulation of RNA polymerase assembly.

How do RPB5 and RMP contribute to cancer progression, and how can antibodies help investigate these mechanisms?

Research indicates that RPB5 and its interacting partner RMP/URI play significant roles in cancer development and progression. Studies focused on hepatocellular carcinoma (HCC) have revealed several important findings:

• RMP expression regulation: RMP expression increases when HCC cells are treated with γ-irradiation, suggesting a role in the cellular response to DNA damage .

• Cell cycle effects: RMP depletion induces G2 arrest of HCC cells, characterized by decreased expression of Cdk1 and Cyclin B .

• Anti-apoptotic function: Cell growth and colony formation assays suggest that RMP plays an anti-apoptotic role in the proliferation and growth of HCC cells .

• In vivo tumor formation: Tumor formation assays confirm the requirement of RMP during HCC growth. Mice injected with cells overexpressing RMP developed larger solid tumors, while no tumors developed in mice injected with RMP-depleted cells .

RPB5 antibodies provide valuable tools for investigating these cancer-related mechanisms:

• Expression analysis: Immunohistochemistry with RPB5 antibodies can assess RPB5 expression patterns across different cancer types and stages. Positive IHC detection of RPB5 has been validated in human breast cancer tissue .

• Protein complex analysis: Co-immunoprecipitation with RPB5 antibodies followed by mass spectrometry can identify cancer-specific protein interactions that may contribute to altered transcriptional regulation.

• ChIP-sequencing: This technique can map genome-wide binding patterns of RNA polymerase II containing RPB5 in cancer cells, potentially revealing cancer-specific transcriptional programs.

• Functional validation: Combining RPB5 antibody-based assays with RMP/URI knockdown or overexpression can elucidate the mechanistic role of the RPB5-RMP interaction in cancer progression.

Additionally, research suggests RMP/URI functions as a transcriptional corepressor for androgen receptor (AR), as depletion of URI enhances AR-mediated gene transcription while overexpression suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth . These findings highlight the potential of targeting the RPB5-RMP axis in cancer therapeutics.

What are the optimal protocols for using RPB5 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) with RPB5 antibodies allows researchers to investigate the genomic binding sites of RNA polymerase II and analyze transcriptional regulation. Based on research protocols and antibody specifications, here are optimized methodologies for RPB5 ChIP experiments:

• Crosslinking optimization:

  • Standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is generally sufficient for RPB5

  • For detecting transient interactions, dual crosslinking with DSG (disuccinimidyl glutarate) before formaldehyde may increase sensitivity

• Chromatin fragmentation:

  • Sonication to generate fragments of 200-500 bp is recommended

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • For RPB5 binding at densely packed chromatin regions, increase sonication time or intensity

• Antibody selection and amount:

  • Use antibodies validated specifically for ChIP applications

  • Optimal antibody amount: 2-5 μg per IP reaction (based on similar nuclear protein ChIP protocols)

  • Consider antibodies targeting different epitopes of RPB5 for validation

• Immunoprecipitation conditions:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Incubate with RPB5 antibody overnight at 4°C with gentle rotation

  • Use BSA-blocked protein A/G beads for capturing antibody-chromatin complexes

• Washing and elution:

  • Use stringent washing buffers with increasing salt concentrations

  • Include LiCl wash to reduce non-specific binding

  • Elute bound chromatin at 65°C in elution buffer containing SDS

• Controls:

  • Input DNA (typically 5-10% of starting chromatin)

  • IgG negative control matched to the host species of RPB5 antibody

  • Positive control: ChIP for well-characterized active gene promoters where RNA polymerase II is known to bind

• Data analysis considerations:

  • Compare RPB5 binding profiles with other RNA polymerase II subunits

  • Correlate with transcriptional activity data

  • Consider sequential ChIP (ChIP-reChIP) with antibodies against other transcription factors to identify co-occupancy

These optimized protocols can facilitate high-quality ChIP data when using RPB5 antibodies, enabling detailed analysis of RNA polymerase II genomic occupancy and transcriptional regulation.

How can RPB5 antibodies be used to investigate interactions with viral proteins like HBx?

The interaction between RPB5 and viral proteins, particularly Hepatitis B virus X protein (HBx), represents an important mechanism by which viruses can modulate host cell transcription. HBx directly interacts with RPB5 and functions as a transcriptional coactivator through this interaction . RPB5 antibodies provide valuable tools for investigating these virus-host interactions:

• Mapping interaction domains:

  • Research has identified four specific sequences in RPB5 (cm70, cm98, cm105, and cm112) that are critical for HBx binding

  • Point mutations at residues V74A, I104A, T111A, and S113A completely abolish HBx-RPB5 interaction

  • RPB5 antibodies can help verify structural integrity of wild-type and mutant RPB5 proteins in these studies

• Competition binding assays:

  • RMP/URI competes with HBx for binding to the same region of RPB5

  • Co-immunoprecipitation with RPB5 antibodies can assess how varying concentrations of HBx affect RMP binding and vice versa

  • This approach helps elucidate the molecular mechanisms of viral transcriptional regulation

• Functional transcription studies:

  • ChIP using RPB5 antibodies can identify genomic regions where RNA polymerase II recruitment is altered by HBx

  • Sequential ChIP (ChIP-reChIP) can determine if HBx and RPB5 co-occupy the same genomic regions

  • These experiments reveal how viral proteins redirect host transcriptional machinery

• Structural analysis:

  • For structural studies of RPB5-HBx complexes, Fab fragments of RPB5 antibodies can stabilize the complex

  • Cryo-EM approaches using RPB5 antibodies as markers can help identify the position of RPB5 within larger complexes

• Therapeutic intervention assessment:

  • RPB5 antibodies can help evaluate potential inhibitors of the RPB5-HBx interaction

  • Monitor changes in RPB5-HBx complex formation using co-IP with RPB5 antibodies in the presence of inhibitor candidates

Understanding the RPB5-HBx interaction provides insights into viral pathogenesis mechanisms and potential therapeutic targets for treating hepatitis B virus-associated diseases, including hepatocellular carcinoma.

What are the best approaches for validating RPB5 antibody specificity in new experimental systems?

Validating antibody specificity is crucial for ensuring reliable and reproducible experimental results. For RPB5 antibodies, comprehensive validation should include multiple complementary approaches:

• Western blot validation:

  • Verify a single band at the expected molecular weight (23-25 kDa)

  • Test in multiple cell lines with known RPB5 expression (positive detection confirmed in A549, HeLa, MCF-7, HepG2, and Jurkat cells)

  • Include negative controls such as lysates from cells with RPB5 knockdown by siRNA or CRISPR

  • For polyclonal antibodies, pre-absorption with the immunizing peptide should eliminate specific bands

• Genetic knockdown/knockout controls:

  • siRNA-mediated knockdown of RPB5 should reduce antibody signal proportionally to protein reduction

  • Inducible expression systems can provide controlled validation of antibody specificity

  • For RNAi experiments targeting RMP/URI, validated constructs include sequences targeting different regions of human RMP (e.g., "GGA UUU GCU AGC UGA UAA ATT")

• Immunoprecipitation validation:

  • IP-Western analysis to confirm antibody pulls down a protein of correct size

  • Mass spectrometry analysis of immunoprecipitated material should identify RPB5 peptides

  • HeLa cells have been validated for positive IP detection of RPB5

• Immunohistochemistry validation:

  • Compare staining patterns with published literature

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive controls (human breast cancer tissue has been validated) and negative controls

• Cross-reactivity assessment:

  • Test with recombinant RPB5 and closely related proteins

  • Evaluate species cross-reactivity if working with non-human models

  • Consider epitope conservation across species when interpreting results

• Application-specific controls:

  • For ChIP applications, include IgG controls and positive controls (known RPB5 binding sites)

  • For fluorescence microscopy, include secondary-only controls and competitive blocking with immunizing peptide

Thorough validation using multiple approaches ensures that experimental results obtained with RPB5 antibodies are specific and reliable, forming a solid foundation for subsequent research.

How can RPB5 antibodies be used to investigate the role of RPB5 in the mTOR signaling pathway?

Research has established intriguing connections between RPB5, its interacting partner RMP/URI, and the mTOR (mammalian Target of Rapamycin) signaling pathway. RMP was originally characterized as a regulator of gene expression controlled by the TOR pathway, and yeast lacking Bud27 (the yeast homolog of RMP/URI) show sensitivity to rapamycin . RPB5 antibodies provide valuable tools for investigating these connections:

• Rapamycin response studies:

  • Research demonstrates that overexpression of RPB5 corrects rapamycin sensitivity in yeast cells lacking Bud27

  • RPB5 antibodies can track changes in RPB5-containing complexes following rapamycin treatment

• Phosphorylation analysis:

  • Evidence indicates that RMP/URI is phosphorylated upon androgen treatment

  • RPB5 antibodies can immunoprecipitate RPB5-containing complexes for subsequent phospho-specific analysis

  • Changes in RPB5 complex composition following mTOR inhibition can reveal regulatory mechanisms

• Transcriptional profiling:

  • ChIP-seq using RPB5 antibodies can identify genomic regions where RNA polymerase II occupancy changes upon mTOR inhibition

  • This approach can help identify mTOR-regulated genes dependent on RPB5-containing complexes

• Protein complex analysis:

  • Co-immunoprecipitation with RPB5 antibodies followed by mass spectrometry can identify changes in RPB5-associated proteins upon mTOR pathway modulation

  • Research has shown that URI/RMP functions as a molecular scaffold assembling a complex of prefoldin proteins

• Subcellular localization:

  • Immunofluorescence with RPB5 antibodies can track changes in localization upon mTOR inhibition

  • This approach can reveal how mTOR signaling affects RNA polymerase complex assembly and nuclear localization

These approaches can help elucidate the molecular mechanisms by which the mTOR pathway regulates transcription through RPB5 and its interacting partners, potentially revealing new therapeutic targets for diseases with dysregulated mTOR signaling, such as cancer and metabolic disorders.