POLR3A Antibody

What is POLR3A and why is it important in cellular function?

POLR3A is the largest subunit of human RNA polymerase III (Pol III), also known as RPC1 or DNA-directed RNA polymerase III subunit A. This protein is critical to the catalytic activity of Pol III as it forms the polymerase active center together with the second largest subunit. Within this complex, a single-stranded DNA template strand of the promoter is positioned within the central active site cleft . POLR3A contributes significantly to Pol III function, which is responsible for transcribing small, untranslated RNAs including 5S rRNA and transfer RNAs (tRNAs) . These RNA molecules are essential for normal cellular functioning and survival, as they play fundamental roles in protein synthesis and other essential cellular processes . Additionally, POLR3A has been identified as part of the innate immune response, functioning as a non-self double-stranded DNA sensor that contributes to detecting and controlling infections by intracellular bacteria and DNA viruses through the production of type I interferons via the RIG-1 pathway . This dual role in both basic transcriptional processes and immune surveillance highlights POLR3A's significance in cellular biology.

What are the key characteristics of available POLR3A antibodies?

Several POLR3A antibodies are commercially available with varying characteristics optimized for different experimental applications. The Proteintech antibody 17530-1-AP is a rabbit polyclonal IgG that targets POLR3A for Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and ELISA applications . This antibody shows reactivity with human, mouse, and rat samples and recognizes POLR3A at an observed molecular weight of 140-150 kDa . Another option, Proteintech's 28842-1-AP, is also a rabbit polyclonal IgG targeting POLR3A, but specifically optimized for WB, IF/ICC, and ELISA applications with confirmed reactivity in human samples only . This antibody detects POLR3A at 155 kDa . Bio-Rad offers the monoclonal antibody clone AB01/1E10, which is a mouse IgG1 that has been purified for enhanced specificity . All these antibodies are supplied in liquid form, typically in PBS buffer with sodium azide and glycerol, and should be stored at -20°C where they remain stable for approximately one year after shipment .

How does POLR3A dysfunction contribute to human disease?

POLR3A dysfunction has been linked to several significant human diseases through genetic studies and functional analyses. Bi-allelic missense variants in the POLR3A gene have been associated with distinct clinical phenotypes including hypogonadotropic hypogonadism and hypomyelinating leukodystrophy with or without oligodontia . Hypomyelinating leukodystrophy is a group of inherited neurodegenerative disorders characterized by abnormal formation or maintenance of myelin in the central nervous system . Research suggests that POLR3A mutations may lead to dysfunction of Pol III, which affects the expression of certain tRNAs that are more abundant in the central nervous system . For example, a recent study demonstrated that the POLR3A p.Cys767Phe variant caused increased expression of mutated POLR3A protein but resulted in abnormal expression of Pol III transcripts and dysfunctional protein activity . This mutation led to decreased expression of Pol III transcripts including BC200 and tRNA Leu-CAA, as well as reduced expression of myelin basic protein (MBP) and 18S rRNA . Additionally, studies have suggested a potential connection between mutated POLR3A and autoimmune diseases such as scleroderma, indicating that POLR3A antibodies may have research applications in investigating autoimmune conditions .

What are the optimal applications for POLR3A antibodies in research?

POLR3A antibodies have been validated across multiple experimental applications, with specific strengths in certain methodologies. Western blotting (WB) is one of the most robustly validated applications, with recommended dilution ranges typically between 1:500-1:2000 for both the 17530-1-AP and 28842-1-AP antibodies . For immunohistochemistry (IHC), the 17530-1-AP antibody has been validated at dilutions between 1:50-1:500, with specific protocols recommending antigen retrieval with TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0 . For immunofluorescence (IF) applications, the 28842-1-AP antibody performs well at dilutions of 1:200-1:800 and has been specifically validated in A431 cells . Both antibodies are also suitable for ELISA applications, and the 17530-1-AP has additionally been validated for immunoprecipitation (IP) . Research publications have confirmed the utility of these antibodies in these applications, though it is recommended that researchers titrate the antibodies in their specific experimental systems to obtain optimal results . When selecting between antibodies, consider the specific cell or tissue type being studied as well as the technical requirements of your experimental approach and the species reactivity needed.

How should researchers optimize Western blot protocols for POLR3A detection?

For optimal Western blot detection of POLR3A, researchers should consider several key methodological factors. First, sample preparation is critical - POLR3A has been successfully detected in various cell lines including HeLa, MCF-7, A431, HEK-293, and Y79 cells . When determining protein concentration, account for POLR3A's large size (observed molecular weight of 140-155 kDa) when selecting gel percentage and run conditions . For gel electrophoresis, an 8-10% SDS-PAGE gel is recommended to properly resolve proteins in this size range. During transfer to membrane, extending transfer time or using specialized protocols for large proteins may improve efficiency. For antibody incubation, start with the manufacturer's recommended dilution range (1:500-1:2000) and optimize through titration experiments . Blocking with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature is generally effective. For primary antibody incubation, overnight incubation at 4°C typically yields the best results, followed by thorough washing steps. When visualizing, both chemiluminescent and fluorescent detection methods are compatible, though exposure times may need optimization given the variable expression levels of POLR3A across different cell types. If background issues arise, increasing wash steps duration and frequency after both primary and secondary antibody incubations can help improve signal-to-noise ratio.

What are the recommended protocols for POLR3A immunofluorescence experiments?

For successful immunofluorescence detection of POLR3A, researchers should follow a carefully optimized protocol. Begin with appropriate fixation - 4% paraformaldehyde for 15-20 minutes at room temperature is generally effective for preserving POLR3A epitopes while maintaining cellular architecture . Permeabilization with 0.2-0.5% Triton X-100 for 5-10 minutes allows antibody access to the nuclear POLR3A. Blocking should be performed with 1-5% normal serum (matched to the species of the secondary antibody) with 0.1-0.3% Triton X-100 in PBS for 1 hour at room temperature. For primary antibody incubation, use the 28842-1-AP antibody at dilutions between 1:200-1:800 in blocking buffer, with overnight incubation at 4°C yielding optimal results . After thorough washing (3-5 times for 5 minutes each with PBS), apply an appropriate fluorophore-conjugated secondary antibody at the manufacturer's recommended dilution for 1-2 hours at room temperature. Following additional washing steps, counterstain nuclei with DAPI or Hoechst and mount with an anti-fade mounting medium. For co-localization studies, POLR3A antibodies can be paired with markers of nuclear transcription factories or other Pol III components. The 28842-1-AP antibody has been specifically validated in A431 cells, providing a positive control for method development . If non-specific background signals occur, additional blocking with 0.1-0.3% BSA or increasing the blocking duration may improve specificity.

How can POLR3A antibodies be utilized in immunohistochemistry of brain tissues?

Immunohistochemical detection of POLR3A in brain tissues requires specialized considerations given the protein's involvement in neurodegenerative conditions such as hypomyelinating leukodystrophy. For formalin-fixed paraffin-embedded (FFPE) brain tissue sections, deparaffinization and rehydration should be followed by antigen retrieval, with the 17530-1-AP antibody specifically recommending either TE buffer at pH 9.0 or citrate buffer at pH 6.0 . Endogenous peroxidase blocking with 3% hydrogen peroxide for 10 minutes helps reduce background. For primary antibody incubation, the 17530-1-AP antibody performs well at dilutions between 1:50-1:500, with overnight incubation at 4°C recommended for brain tissues . Detection systems utilizing HRP-conjugated secondary antibodies followed by DAB chromogen development provide good visualization of POLR3A expression patterns. The antibody has been positively validated in mouse cerebellum tissue, making this an appropriate positive control . When examining brain sections, particular attention should be paid to white matter regions and oligodendrocytes, as POLR3A mutations are associated with hypomyelination disorders. For co-localization studies in brain tissue, dual immunofluorescence with myelin markers such as MBP can provide valuable insights into how POLR3A dysfunction may affect myelination processes . Careful titration is essential when working with brain tissues, as expression levels may vary between different neural cell populations and across developmental stages.

How can researchers address common challenges in POLR3A antibody experiments?

Researchers frequently encounter several challenges when working with POLR3A antibodies that can be systematically addressed. For weak or absent signals in Western blots, first verify POLR3A expression in your sample type, as expression levels vary across tissues and cell lines. The antibodies have been validated in HeLa and MCF-7 cells, which can serve as positive controls . If signal remains weak, increasing protein loading, extending primary antibody incubation time, or using enhanced chemiluminescence detection systems can improve sensitivity. For high background issues, optimize blocking conditions (consider 5% BSA instead of milk for phosphorylation studies), increase wash duration and frequency, and ensure antibody dilutions are appropriate. If multiple bands appear in Western blots, verify that you're examining the correct molecular weight range (140-155 kDa) and consider that post-translational modifications or splice variants might be present . For immunostaining applications showing non-specific signals, increase blocking duration, optimize antibody concentration through careful titration, and consider including additional blocking agents such as 0.1-0.3% BSA. If fixation-dependent artifacts occur in immunofluorescence, compare 4% paraformaldehyde with methanol fixation to determine which better preserves your epitope. For inconsistent results between experiments, standardize protocols rigorously and prepare single-use aliquots of antibodies to avoid freeze-thaw cycles that can degrade antibody quality over time.

What controls should be included in POLR3A antibody experiments?

Rigorous experimental design for POLR3A antibody applications requires appropriate controls to ensure reliable and interpretable results. Positive controls should include cell lines or tissues with confirmed POLR3A expression. According to validation data, HeLa cells, MCF-7 cells, A431 cells, HEK-293 cells, and Y79 cells have demonstrated positive POLR3A expression in Western blots . For tissue sections, mouse cerebellum has been validated for IHC applications . Negative controls are equally crucial: for primary antibody validation, include a sample incubated with isotype-matched IgG instead of the POLR3A antibody. For more stringent validation, POLR3A knockdown or knockout cells provide excellent negative controls by demonstrating specificity of the antibody signal. Loading controls for Western blots should include housekeeping proteins of different molecular weights than POLR3A (156 kDa) to avoid overlap issues. Technical replicates (minimum of three) are necessary to establish reproducibility of findings. For cellular localization studies, nuclear counterstaining is essential since POLR3A is primarily localized to the nucleus . When investigating POLR3A mutations or variants, wild-type expression systems provide critical comparison points, as demonstrated in studies examining the functional consequences of the p.Cys767Phe variant, where both wild-type and mutant POLR3A were overexpressed for comparative analysis .

How do POLR3A protein modifications affect antibody recognition?

POLR3A protein can undergo various post-translational modifications that potentially affect antibody recognition and experimental outcomes. While the primary calculated molecular weight of POLR3A is 156 kDa based on its 1390 amino acid sequence, observed molecular weights in SDS-PAGE typically range from 140-155 kDa, suggesting some variability in protein processing or detection . Researchers should be aware that phosphorylation states may influence antibody binding, particularly for antibodies raised against epitopes containing phosphorylation sites. Although specific phosphorylation information for these commercial antibodies is not detailed in the search results, it remains an important consideration for experimental design. For the 17530-1-AP antibody, the immunogen used was a POLR3A fusion protein (Ag11536), while the 28842-1-AP antibody utilized a different fusion protein (Ag29036) . These different immunogens might recognize distinct epitopes on the POLR3A protein, potentially explaining any differences in detection patterns between the antibodies. When investigating mutant forms of POLR3A, such as the p.Cys767Phe variant, researchers should verify whether their antibody's epitope overlaps with or is affected by the mutation site . If studying disease-associated variants, it may be beneficial to test multiple POLR3A antibodies targeting different epitopes to ensure comprehensive detection of both wild-type and mutant proteins, as mutations might alter protein conformation and epitope accessibility.

How can POLR3A antibodies contribute to leukodystrophy research?

POLR3A antibodies offer valuable tools for investigating the molecular mechanisms underlying POLR3A-related hypomyelinating leukodystrophy. These antibodies enable researchers to examine POLR3A expression patterns in normal and pathological brain tissues, particularly focusing on white matter regions and oligodendrocytes responsible for myelin production . Immunohistochemistry using the 17530-1-AP antibody, which has been validated in mouse cerebellum tissue, can reveal the distribution of POLR3A in brain regions affected by leukodystrophy . Western blot analysis comparing POLR3A protein levels between patient-derived samples and controls can identify alterations in protein expression or molecular weight that might indicate pathogenic variants. Co-immunoprecipitation experiments using POLR3A antibodies can identify interacting proteins that may be disrupted in disease states. For functional studies, researchers can use cellular models expressing POLR3A mutations identified in patients (such as the p.Cys767Phe variant) and employ POLR3A antibodies to assess protein expression, subcellular localization, and association with Pol III components . Critically, these antibodies enable assessment of how POLR3A mutations affect transcription of specific Pol III targets like 5S rRNA and tRNAs that may be particularly important in neural cells. As demonstrated in recent research, comparative analysis of wild-type versus mutant POLR3A overexpression systems can elucidate how specific mutations affect Pol III transcriptional activity and downstream targets like MBP, which is essential for proper myelination .

What methodologies can assess POLR3A's role in immune response pathways?

POLR3A plays a significant role in innate immunity as a non-self double-stranded DNA sensor, contributing to the detection and control of intracellular bacteria and DNA viruses . Researchers investigating this immunological function can employ several antibody-dependent methodologies. Co-immunoprecipitation using POLR3A antibodies can identify interactions with immune signaling components following pathogen exposure. Chromatin immunoprecipitation (ChIP) assays can determine whether POLR3A binding to chromatin is altered during immune responses or by pathogen-derived molecules. For cellular localization studies, immunofluorescence with the 28842-1-AP antibody (dilution 1:200-1:800) can track POLR3A redistribution following immune stimulation . To investigate POLR3A's role in the RIG-I pathway and type I interferon production, researchers can combine POLR3A immunoprecipitation with RNA immunoprecipitation (RIP) to identify RNAs associated with POLR3A during immune activation . Western blot analysis using the validated antibodies can assess POLR3A protein levels and post-translational modifications following exposure to pathogen-associated molecular patterns or during viral infection . Flow cytometry with permeabilization protocols can quantify POLR3A levels across immune cell populations when appropriately validated. For functional studies, combining gene editing approaches (CRISPR/Cas9) targeting POLR3A with antibody-based detection methods can establish causal relationships between POLR3A activity and specific immune responses. These methodologies collectively enable detailed characterization of how POLR3A contributes to pathogen sensing and innate immune signaling pathways.

How can POLR3A antibodies be used in studying Pol III transcription dynamics?

POLR3A antibodies provide powerful tools for investigating the complex dynamics of RNA polymerase III-mediated transcription. Chromatin immunoprecipitation (ChIP) assays using POLR3A antibodies enable mapping of Pol III occupancy across the genome, identifying active transcription sites for tRNAs, 5S rRNA, and other Pol III targets. For studying transcription complex assembly, co-immunoprecipitation with POLR3A antibodies can identify interacting components and regulatory factors that associate with the Pol III complex under different cellular conditions. Researchers investigating transcriptional responses to cellular stresses or signaling events can use Western blotting with validated POLR3A antibodies (recommended dilutions 1:500-1:2000) to monitor changes in POLR3A protein levels . Immunofluorescence microscopy using the 28842-1-AP antibody allows visualization of POLR3A nuclear distribution and potential colocalization with transcription factories or specific genomic loci when combined with FISH techniques . For kinetic studies of transcription, combining POLR3A immunoprecipitation with nascent RNA sequencing can reveal real-time Pol III activity across the genome. When investigating disease-associated POLR3A variants, researchers can compare wild-type and mutant POLR3A proteins regarding their association with chromatin and transcriptional output, as demonstrated in functional studies of the p.Cys767Phe variant . These approaches have revealed that mutant POLR3A proteins, despite sometimes showing increased expression levels, may exhibit frustrated Pol III transcription function with decreased expression of targets like BC200, tRNA Leu-CAA, and downstream effects on genes like MBP . Such methodologies contribute to understanding both basic Pol III biology and the pathological consequences of POLR3A dysfunction.

What are emerging applications for POLR3A antibodies in research?

Emerging applications for POLR3A antibodies include several cutting-edge research directions exploiting advances in both antibody technology and research methodologies. Single-cell approaches combining POLR3A antibodies with single-cell RNA sequencing can reveal cell-type-specific roles of POLR3A in heterogeneous tissues, particularly within the complex cellular environment of the brain where POLR3A mutations cause hypomyelinating leukodystrophy . Super-resolution microscopy techniques utilizing validated POLR3A antibodies for immunofluorescence (such as 28842-1-AP at dilutions of 1:200-1:800) can provide unprecedented insights into the nuclear organization of Pol III transcription factories and how they interact with chromatin domains . For translational applications, POLR3A antibodies may facilitate development of diagnostic tools for leukodystrophies and autoimmune conditions like scleroderma where POLR3A has been implicated . The generation of phospho-specific POLR3A antibodies represents another frontier, enabling researchers to track how post-translational modifications regulate Pol III activity in response to cellular signaling or stress conditions. CRISPR-engineered cell lines expressing tagged POLR3A variants combined with validated antibodies can create powerful tools for studying POLR3A dynamics in living cells. As therapeutic approaches targeting RNA polymerases evolve, POLR3A antibodies will play crucial roles in validating target engagement and specificity. The continuing development of antibodies with enhanced specificity for different POLR3A epitopes will further expand research capabilities, particularly for distinguishing between wild-type and mutant forms of POLR3A that may coexist in cells heterozygous for disease-causing variants .

How should researchers interpret discrepancies in POLR3A antibody results?

When researchers encounter discrepancies in results obtained with different POLR3A antibodies, a systematic approach to troubleshooting and interpretation is essential. First, consider the antibodies' target epitopes - the 17530-1-AP and 28842-1-AP antibodies were raised against different fusion protein immunogens (Ag11536 and Ag29036, respectively), which may recognize distinct regions of the POLR3A protein . If conflicting results occur in protein size detection (the observed molecular weight ranges from 140-155 kDa across different antibodies), this could reflect genuine biological variations such as alternative splicing, post-translational modifications, or tissue-specific processing . Species-specific differences may also contribute to discrepancies, as the 17530-1-AP antibody shows reactivity with human, mouse, and rat samples, while the 28842-1-AP is validated only for human samples . Technical factors such as sample preparation methods, buffer compositions, fixation protocols for immunostaining, and detection systems can significantly impact results. When investigating disease-associated variants such as the p.Cys767Phe mutation, consider whether the mutation affects the antibody's epitope or alters protein conformation, potentially affecting recognition . To resolve discrepancies, researchers should directly compare antibodies under identical experimental conditions, include appropriate positive and negative controls, and validate findings using complementary approaches such as gene expression analysis or functional assays measuring Pol III activity. When publishing potentially conflicting results, transparent reporting of the specific antibody clone, catalog number, dilution, and detailed methodologies is essential to enable proper interpretation and reproducibility by the broader scientific community.

What future developments in POLR3A antibody technology might advance the field?

Future advancements in POLR3A antibody technology will likely expand research capabilities and clinical applications in several directions. Development of conformation-specific antibodies could distinguish between active and inactive forms of POLR3A, providing insights into the catalytic dynamics of Pol III. Engineered recombinant antibodies with enhanced specificity for particular POLR3A domains will improve signal-to-noise ratios and detection sensitivity, especially for low-abundance forms of the protein. Nanobodies derived from camelid antibodies offer advantages of smaller size and potentially better access to cryptic epitopes within large protein complexes like Pol III, opening new possibilities for structural and functional studies. For clinical applications, development of antibodies specifically recognizing common pathogenic POLR3A variants (such as those causing hypomyelinating leukodystrophy) could facilitate diagnostic testing and patient stratification . Multiplexed antibody technologies combining POLR3A detection with other Pol III components or downstream targets could enable comprehensive pathway analysis in limited biological samples. For functional studies, intrabodies designed for expression in living cells could track POLR3A dynamics in real-time without fixation artifacts. Antibody fragments optimized for super-resolution microscopy techniques will advance our understanding of POLR3A's nuclear organization and interactions. Development of species-specific antibodies with cross-species reactivity validation will enhance comparative studies across model organisms. As therapeutic approaches targeting transcriptional machinery evolve, antibodies with high specificity for POLR3A could facilitate the development of antibody-drug conjugates or targeted protein degradation strategies for conditions where aberrant POLR3A activity contributes to pathology.

What cell lines and tissue samples are optimal for POLR3A antibody validation?

Optimal validation of POLR3A antibodies requires careful selection of appropriate cell lines and tissue samples based on documented expression patterns and experimental needs. For cell line validation, several human cell lines have been positively tested and can serve as reliable positive controls. HeLa and MCF-7 cells have been validated for both the 17530-1-AP and 28842-1-AP antibodies in Western blot applications . Additional human cell lines showing positive Western blot results with the 28842-1-AP antibody include A431, HEK-293, and Y79 cells . For immunofluorescence/immunocytochemistry validation, A431 cells have been specifically validated with the 28842-1-AP antibody . For tissue validation, mouse cerebellum tissue has been confirmed as positive for POLR3A expression using immunohistochemistry with the 17530-1-AP antibody . When investigating neurological conditions related to POLR3A mutations, brain tissue samples, particularly white matter regions, are highly relevant given POLR3A's role in hypomyelinating leukodystrophies . For researchers developing new applications, comparing antibody performance across multiple validated cell lines provides robust quality control. When selecting validation samples for disease-related research, consider including both normal and patient-derived materials when available, particularly for studies of leukodystrophy or autoimmune conditions where POLR3A has been implicated . For cross-species studies, it's important to note that while the 17530-1-AP antibody has been validated in human, mouse, and rat samples, the 28842-1-AP antibody has only been confirmed in human samples . This species-specific validation information is crucial when designing experiments using animal models of POLR3A-related diseases.

What quantification methods are appropriate for POLR3A expression analysis?

Accurate quantification of POLR3A expression requires selection of appropriate methodologies based on experimental goals and sample characteristics. For Western blot quantification, densitometric analysis of POLR3A bands (observed at 140-155 kDa) should be normalized to appropriate loading controls, avoiding housekeeping proteins of similar molecular weight . Digital imaging systems with linear dynamic range are preferable to film for quantitative Western blotting. When developing quantification protocols, researchers should establish standard curves using recombinant POLR3A protein to confirm linearity of detection within their working range. For immunohistochemical quantification, considerations include the analysis of staining intensity (using calibrated optical density measurements), percentage of positive cells, and scoring systems that combine both parameters. Digital pathology approaches using validated image analysis algorithms can reduce subjective interpretation and increase reproducibility. For immunofluorescence quantification, measuring nuclear fluorescence intensity of POLR3A relative to DAPI or other nuclear markers provides normalized expression data, ideally using confocal microscopy to ensure accurate subcellular localization . When comparing wild-type and mutant POLR3A expression, as in studies of the p.Cys767Phe variant, parallel quantification of both protein levels and functional outputs (such as Pol III transcripts like 5S rRNA, BC200, and tRNA Leu-CAA) provides comprehensive assessment . Flow cytometry with permeabilization protocols offers high-throughput quantification across cell populations when antibodies are appropriately validated for this application. For all quantification approaches, technical replicates (minimum of three) and appropriate statistical analysis are essential, with careful consideration of data distribution properties when selecting parametric versus non-parametric statistical tests.