POLR1C Antibody

What applications are POLR1C antibodies validated for, and what are the optimal working dilutions?

POLR1C antibodies have been validated for multiple applications with specific recommended dilutions:

When optimizing these applications, it's critical to titrate the antibody concentration in your specific experimental system. Different cell lines may require adjusted concentrations based on expression levels. For Western blotting, the predicted molecular weight of POLR1C is 39 kDa, which should be confirmed in your experimental system . For immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative .

What is the molecular function of POLR1C protein, and why is it important for researchers to study?

POLR1C (DNA-directed RNA polymerases I and III subunit RPAC1) functions as a common component of both RNA polymerase I and III complexes. These polymerases are essential for:

• RNA polymerase I: Synthesizes ribosomal RNA (rRNA) precursors

• RNA polymerase III: Produces short non-coding RNAs including 5S rRNA, snRNAs, tRNAs, and miRNAs

Within the polymerase core, POLR1C appears to function as a clamp element that regulates the opening and closing of the central cleft, controlling access to the catalytic site . This dual role in two distinct polymerase complexes makes POLR1C particularly interesting for studying transcriptional regulation and ribosome biogenesis.

Research importance stems from its involvement in neurodevelopmental disorders (Treacher Collins syndrome, hypomyelinating leukodystrophy) and potential role in fundamental processes like nucleolar stress and aging .

How can researchers validate the specificity of commercial POLR1C antibodies in their experimental system?

Establishing antibody specificity requires multiple validation approaches:

• Positive control validation: Use cell lines with confirmed POLR1C expression. BxPC-3, HEK-293, Caco-2, HeLa, and HepG2 cells have been validated for Western blot applications .

• Cross-species validation: POLR1C is highly conserved, allowing antibody testing across human and mouse samples. Validated antibodies show consistent detection at the predicted molecular weight (39 kDa) .

• Knockout/knockdown controls: Compare antibody signal in POLR1C knockout or siRNA-treated samples versus wildtype. A specific antibody should show significantly reduced signal.

• Multiple antibody validation: Use antibodies targeting different POLR1C epitopes. For example, compare antibodies targeting the C-terminal region (such as AMS.AP2839b) with those targeting amino acids 50-200 (such as ab246976) .

• Immunoprecipitation-mass spectrometry: Perform IP followed by mass spectrometry to confirm that the immunoprecipitated protein is indeed POLR1C.

When publishing results, include detailed validation data and specify the exact antibody clone, dilution, and incubation conditions to ensure reproducibility.

What are the key considerations for optimizing immunofluorescence protocols with POLR1C antibodies?

For successful immunofluorescence using POLR1C antibodies:

How can POLR1C antibodies be used to investigate mechanisms of hypomyelinating leukodystrophy?

POLR1C antibodies offer critical insights into hypomyelinating leukodystrophy pathomechanisms:

• Aberrant splicing detection: POLR1C variants cause abnormal splicing with intron inclusion in 85% of transcripts. Design experiments using splicing-specific antibodies alongside standard POLR1C antibodies to track both normal and aberrant protein products .

• Differential subcellular localization: Compare POLR1C localization in patient-derived cells versus controls using immunofluorescence. POLR1C variants can cause altered protein subcellular localization, as demonstrated in functional analyses of biallelic POLR1C alterations (c.167T>A, p.M56K and c.595A>T, p.I199F) .

• Protein-protein interaction studies: Use POLR1C antibodies for co-immunoprecipitation to assess how mutations affect interactions with other RNA polymerase subunits.

• Expression level quantification: Western blot analysis with POLR1C antibodies reveals decreased protein expression in patient cells, correlating with disease severity .

• Tissue-specific investigations: Immunohistochemistry in brain tissue can map POLR1C expression patterns in regions associated with myelin production.

These approaches help establish connections between genetic variants, protein dysfunction, and cellular phenotypes in leukodystrophy research.

What role can POLR1C antibodies play in investigating Treacher Collins syndrome pathogenesis?

POLR1C antibodies are valuable tools for studying Treacher Collins syndrome (TCS) mechanisms:

• Nucleolar localization analysis: TCS-specific POLR1C variants disrupt protein localization in the nucleolus, primarily affecting Pol I function rather than Pol III . Immunofluorescence with POLR1C antibodies can track this mislocalization.

• Quantitative assessment of subunit incorporation: Western blot analyses of nuclear versus nucleolar fractions can determine how TCS-related mutations affect POLR1C incorporation into polymerase complexes.

• Craniofacial development studies: Immunohistochemistry in developmental tissue can track POLR1C expression in neural crest cells critical for craniofacial formation .

• Transcriptional activity correlation: Combine POLR1C immunofluorescence with nascent RNA labeling to correlate POLR1C localization with transcriptional output in TCS models.

• Interaction with Treacle: Co-immunoprecipitation using POLR1C antibodies can determine how TCS mutations affect interactions with Treacle (TCOF1), which forms a complex with UBF, SL1, and Pol I, functioning in rDNA transcription .

Research indicates that while POLR1C is part of both Pol I and Pol III, TCS pathogenesis primarily involves Pol I dysfunction, highlighting the importance of examining nucleolar-specific activities .

How can POLR1C antibodies be utilized to investigate RNA polymerase I as a target for neurodegenerative disorders?

Recent research identifies RNA polymerase I as a potential therapeutic target for neurodegenerative disorders characterized by abnormal protein accumulation:

• Nucleolar stress detection: Use POLR1C antibodies to monitor nucleolar morphology and composition in neurodegenerative models. Changes in POLR1C localization can indicate nucleolar stress, which has been linked to neuronal death in various disorders .

• Polymerase I inhibition studies: In experiments exploring Pol I inhibition as a therapeutic strategy, POLR1C antibodies can confirm target engagement. Western blot and immunofluorescence can monitor POLR1C levels and localization during drug treatment .

• Cell-type specific responses: Immunohistochemistry with POLR1C antibodies in brain tissue can identify differential vulnerability of neuronal populations to polymerase dysfunction.

• Ribosome biogenesis monitoring: As POLR1C is essential for rRNA transcription, antibodies against it can track ribosome biogenesis in response to therapies targeting protein synthesis rates in protein accumulation disorders .

• Therapeutic window determination: Quantitative analysis of POLR1C levels can help determine:

  • How much Pol I inhibition neurons can tolerate

  • Whether inhibition should be continuous or pulsed

  • If cells compensate for Pol I inhibition by upregulating active rDNAs

These approaches provide critical insights into the potential of Pol I as a therapeutic target for conditions like Alzheimer's disease, α-synucleinopathies, and tauopathies.

What methodological approaches can be used to investigate dysregulation of splicing in POLR1C-related disorders?

Investigating splicing dysregulation in POLR1C-related disorders requires sophisticated methodological approaches:

• Long-read sequencing: As demonstrated in research on POLR1C variants, long-read sequencing (e.g., Nanopore MinION) can detect and quantify complex splicing events including intron retention. This approach revealed that 85% of POLR1C transcripts in patient cells contained abnormal intron inclusions .

• Allele-specific analysis: Design experiments to track splicing on both mutant and wild-type alleles, as POLR1C variants unexpectedly caused intron inclusions on both alleles in heterozygous carriers .

• Antibody selection for splice variant detection: Use antibodies targeting regions present in both normal and mis-spliced variants to quantify total protein, combined with splice junction-specific antibodies to differentiate between isoforms.

• Temporal dynamics assessment: Pulse-chase experiments with POLR1C antibodies can track protein turnover rates of normally spliced versus aberrantly spliced variants.

• Downstream target analysis: As POLR1C splicing defects may affect other genes, expand investigations beyond POLR1C itself to examine global splicing dysregulation patterns.

The discovery that POLR1C variants cause widespread splicing abnormalities suggests a potential downstream pathomechanism rather than direct consequences of the variants themselves, highlighting the importance of comprehensive splicing assessment in these disorders .

What is the significance of POLR1C in regulation of transcription, and how can antibodies help elucidate its regulatory mechanisms?

POLR1C serves as a crucial regulatory component in transcriptional control through several mechanisms:

• Dual polymerase functionality: POLR1C participates in both RNA polymerase I (nucleolar, rRNA synthesis) and RNA polymerase III (nuclear, tRNA and small RNA synthesis) complexes. This dual role positions it as a potential coordinator of different RNA production pathways . Antibodies targeting POLR1C can be used in ChIP-seq experiments to map genome-wide binding patterns of both polymerases.

• Clamp function in polymerase machinery: Within the polymerase core, POLR1C functions as a clamp element that regulates opening and closing of the central cleft, controlling access to the catalytic site . Structure-function studies using antibodies can help determine how this mechanical function varies between different polymerase complexes.

• Response to regulatory signals: POLR1C is subject to regulation by factors like MAF1, p53, and RB1 . Co-immunoprecipitation experiments with POLR1C antibodies can identify:

  • How these regulatory interactions differ between cell types

  • How stress conditions modify these interactions

  • Which regulatory pathways predominate in different developmental contexts

• Nucleolar stress response: POLR1C localization changes during nucleolar stress, which has been linked to aging and neurodegeneration . Time-course immunofluorescence studies with POLR1C antibodies can track dynamic responses to various stressors.

• Developmental regulation: POLR1C expression patterns change during development, particularly in tissues affected by POLR1C-related disorders . Immunohistochemistry with POLR1C antibodies in developmental tissue series can map these critical transitions.

Understanding these regulatory mechanisms provides insights into both basic transcriptional control and disease pathogenesis in POLR1C-related disorders.

What are common sources of variability when using POLR1C antibodies, and how can researchers address them?

Several factors can introduce variability in POLR1C antibody experiments:

• Antibody epitope location: Different POLR1C antibodies target distinct regions of the protein:

  • Ab246976: amino acids 50-200

  • AP2839b: C-terminal region

  • EPR16178: unspecified epitope, but monoclonal

  Solution: When comparing data across studies or during experimental troubleshooting, consider epitope differences which may affect detection of specific protein conformations or complexes.

• Cell type-specific expression: POLR1C expression varies across cell types, with confirmed expression in:

  • Human: A431, BxPC-3, HEK-293, Caco-2, HeLa, HepG2 cells

  • Mouse: NIH/3T3 cells

  Solution: Include positive control cell lines with validated expression when testing a new system.

• Nucleolar integrity: As POLR1C localizes to nucleoli, procedures that disrupt nucleolar integrity can affect detection.

  Solution: Preserve nucleolar structure by using gentle fixation protocols and avoid harsh extraction procedures for nuclear proteins.

• Post-translational modifications: These may affect epitope accessibility.

  Solution: Consider using multiple antibodies targeting different regions to ensure comprehensive detection.

• Splice variants and mutations: POLR1C variants can cause abnormal splicing with intron retention .

  Solution: Design experiments to account for potential splice variants, particularly when studying disease models.

By systematically addressing these variables, researchers can improve reproducibility and data interpretation in POLR1C studies.

How should researchers approach contradictory results between different POLR1C antibodies or detection methods?

When faced with contradictory results between different POLR1C antibodies or methods:

• Comprehensive antibody validation: Return to fundamental validation steps for each antibody:

  • Western blot confirmation of predicted 39 kDa band

  • Signal reduction in POLR1C knockdown/knockout samples

  • Cross-validation with multiple antibodies targeting different epitopes

  • Mass spectrometry confirmation of immunoprecipitated protein identity

• Methodological cross-validation: Compare results across multiple techniques:

  • If immunofluorescence shows nuclear localization but fractionation studies show cytoplasmic presence, consider potential nuclear envelope disruption during fractionation

  • If Western blot and immunofluorescence yield different results, investigate fixation conditions and detergent sensitivity

• Epitope mapping analysis: Consider whether discrepancies might reflect detection of different protein states:

  • Antibodies recognizing distinct epitopes might differentially detect POLR1C in complex with Pol I versus Pol III

  • Some epitopes might be masked when POLR1C is incorporated into polymerase complexes

• Literature reconciliation: Compare conditions with published studies:

  • The Proteintech antibody (15923-1-AP) has been cited in mouse studies

  • EPR16178 has been validated in rat, human, and mouse samples

  • Understanding species-specific variations might explain discrepancies

• Multiparametric approach: Implement integrative approaches combining:

  • Genomic methods (ChIP-seq)

  • Transcriptomic methods (RNA-seq)

  • Proteomic methods (IP-MS)

  • Localization studies (IF/IHC)