POLRMT Antibody, HRP conjugated

What is POLRMT and why is it significant in research?

POLRMT (mitochondrial RNA polymerase) is responsible for transcribing the mitochondrial genome that encodes essential components of oxidative phosphorylation. Recent studies have identified POLRMT as a potential oncogene, with significant overexpression observed in multiple cancer types including skin squamous cell carcinoma (SCC), non-small cell lung cancer (NSCLC), and osteosarcoma . This makes POLRMT a compelling research target for understanding mitochondrial dysfunction in cancer.

The protein functions as the primary RNA polymerase in mitochondria, transcribing genes that encode critical respiratory chain complex subunits. Experimental evidence indicates that genetic depletion of POLRMT through shRNA knockdown or CRISPR/Cas9-mediated knockout significantly impairs cancer cell viability, proliferation, and migration while inducing apoptosis . These findings establish POLRMT as both a valuable biomarker and potential therapeutic target in cancer research.

What applications is the POLRMT Antibody, HRP conjugated suitable for?

• Western Blotting: The HRP conjugation eliminates the need for secondary antibody incubation, potentially reducing background signal and streamlining protocols. This application is particularly valuable when analyzing POLRMT expression levels in different cell types or experimental conditions.

• Immunohistochemistry (IHC): While not explicitly tested according to the product information, HRP-conjugated antibodies are frequently employed in IHC applications. Researchers investigating POLRMT expression in tissue samples should conduct pilot experiments to optimize antigen retrieval methods and antibody dilutions.

• Multiplexed Immunoassays: The direct HRP conjugation facilitates inclusion in multiplexed detection systems where multiple targets are analyzed simultaneously.

When developing new applications, preliminary validation is essential, as antibody performance can vary between experimental systems.

What are the optimal storage and handling conditions for POLRMT Antibody, HRP conjugated?

Proper storage and handling are critical for maintaining antibody activity and experimental reproducibility. The POLRMT Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . Researchers should note these specific considerations:

• Aliquoting: To minimize freeze-thaw cycles, divide the antibody into single-use aliquots upon receipt. HRP conjugation makes the antibody particularly susceptible to activity loss with repeated freeze-thaw cycles.

• Buffer Composition: The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), with 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during storage.

• Working Dilution Preparation: When preparing working dilutions, use freshly prepared, cold buffer and maintain the antibody at 4°C during experimental procedures to preserve HRP activity.

• Avoiding Contamination: Use sterile technique when handling to prevent microbial contamination that could degrade the antibody or interfere with experimental results.

• Light Sensitivity: Minimize exposure to bright light as HRP conjugates can be photosensitive.

Following these handling guidelines will maximize antibody performance and experimental reproducibility.

How can researchers validate POLRMT Antibody specificity for their experimental systems?

Antibody validation is essential for ensuring experimental rigor. For POLRMT Antibody, researchers should implement a multi-pronged validation approach:

• Genetic Knockdown/Knockout Controls: Generate POLRMT-depleted samples using shRNA or CRISPR/Cas9 methods as described in multiple studies . These provide definitive negative controls for antibody specificity assessment.

• Western Blot Analysis: Researchers should be aware that some POLRMT antibodies may detect a non-specific cross-reacting band that migrates above the POLRMT protein . Compare patterns between wild-type and POLRMT-depleted samples to identify specific bands.

• Immunoprecipitation Followed by Mass Spectrometry: This approach can confirm that the antibody is capturing the intended target protein.

• Recombinant Protein Testing: Use purified recombinant POLRMT protein as a positive control to establish detection sensitivity and specificity.

• Tissue/Cell Type Comparison: Analyze samples with known differential expression of POLRMT. For instance, compare normal skin cells to SCC cells, where POLRMT is significantly elevated .

This comprehensive validation strategy ensures reliable experimental outcomes and facilitates accurate interpretation of results.

What methodological approaches can be employed to investigate POLRMT's role in cancer using the antibody?

Researchers can employ several strategic approaches to study POLRMT's oncogenic functions:

• Expression Correlation Studies: Use the POLRMT Antibody in immunoblotting or immunohistochemistry to quantify POLRMT levels across cancer stages. Research has demonstrated that POLRMT is significantly elevated (approximately three-fold) in skin SCC tumor tissues compared to normal skin tissues .

• Functional Consequence Analysis: After POLRMT manipulation (knockdown/overexpression), analyze:

  • Mitochondrial transcript levels (NDUFB8, UQCRC2, COXI)

  • Mitochondrial membrane potential using JC-1 or TMRM dyes

  • ROS production via fluorescent probes

  • ATP production

  • Apoptotic markers (TUNEL assay, caspase activity)

• Therapeutic Target Validation: Combine POLRMT antibody-based detection with IMT1 (POLRMT inhibitor) treatment to correlate POLRMT expression levels with inhibitor sensitivity.

• In Vivo Xenograft Models: Use the antibody to monitor POLRMT expression in xenograft tissues after interventions. Studies have shown that POLRMT shRNA adeno-associated virus injection robustly hindered growth of subcutaneous A431 xenografts in mice .

These approaches can help establish the mechanistic role of POLRMT in cancer progression and evaluate its potential as a therapeutic target.

How can researchers quantitatively assess mitochondrial transcription activity in relation to POLRMT expression?

To establish functional relationships between POLRMT levels and mitochondrial transcription activity, researchers should implement these methodological approaches:

• Mitochondrial Transcript Quantification: Use quantitative RT-PCR to measure levels of mitochondria-encoded transcripts that are directly dependent on POLRMT activity, specifically NDUFB8, UQCRC2, and COXI . This provides a direct assessment of POLRMT transcriptional output.

• mtDNA Content Analysis: Quantify mitochondrial DNA copy number using qPCR and correlate with POLRMT protein levels determined by the antibody. Research has shown decreased mtDNA content in POLRMT-silenced or knockout NSCLC cells .

• Respiratory Chain Complex Assembly: Use Blue Native PAGE combined with western blotting to evaluate how POLRMT expression impacts the assembly of respiratory chain complexes.

• Nascent RNA Synthesis Measurement: Employ BrU incorporation assays specifically in mitochondria to directly measure the rate of mitochondrial transcription in relation to POLRMT levels.

• Correlation Analysis: Generate quantitative data sets that correlate POLRMT protein levels (using the HRP-conjugated antibody) with transcriptional output measurements across different experimental conditions.

This multifaceted approach enables researchers to establish causal relationships between POLRMT expression and mitochondrial transcriptional activity.

What considerations should be made when designing experiments to detect POLRMT in complex tissue samples?

Detecting POLRMT in complex tissue samples presents several technical challenges that require careful experimental design:

• Tissue Preparation and Fixation: Different fixation methods can affect epitope accessibility. For POLRMT detection in tissues, researchers should compare multiple fixation protocols (formalin, paraformaldehyde, alcohol-based) to determine optimal antigen preservation.

• Antigen Retrieval Optimization: Test various antigen retrieval methods, including heat-induced epitope retrieval at different pH values and enzymatic retrieval approaches, to maximize POLRMT detection while maintaining tissue morphology.

• Signal Amplification Systems: For tissues with lower POLRMT expression, consider implementing tyramide signal amplification systems compatible with HRP-conjugated antibodies to enhance detection sensitivity.

• Multiplexed Analysis: Design co-staining experiments to examine POLRMT in relation to cell type-specific markers, mitochondrial markers, or cancer markers. This requires careful selection of compatible antibodies and fluorophores.

• Quantitative Image Analysis: Develop robust image analysis workflows for quantifying POLRMT staining intensity relative to appropriate controls across tissue regions.

• Validation with Molecular Methods: Confirm immunohistochemical findings with molecular techniques such as laser capture microdissection followed by RT-qPCR or western blotting.

This comprehensive approach enables reliable detection and quantification of POLRMT in heterogeneous tissue samples.

How can POLRMT Antibody be used to investigate interactions between POLRMT and other mitochondrial transcription factors?

POLRMT functions within a complex transcriptional machinery that includes multiple factors. To investigate these interactions:

• Co-Immunoprecipitation Studies: Use the POLRMT antibody (non-HRP conjugated version) to immunoprecipitate POLRMT and associated proteins. Western blot analysis can then detect co-precipitated factors such as TFAM and TFB2M .

• Proximity Ligation Assay (PLA): This technique can visualize and quantify protein-protein interactions within cells. Researchers can use POLRMT antibody in combination with antibodies against other transcription factors to detect specific interactions at the single-molecule level.

• ChIP-seq Applications: Although challenging with mitochondrial proteins, chromatin immunoprecipitation followed by sequencing can map POLRMT binding sites on mtDNA and identify co-binding with other factors.

• Protein Complex Analysis: Blue Native PAGE followed by western blotting can identify native protein complexes containing POLRMT and other transcription components.

Research has shown that POLRMT interacts with MRPL12, which plays a role in its function . These methodological approaches can further elucidate the complex interactions governing mitochondrial transcription.

What are the most effective approaches for monitoring POLRMT dynamics during cellular stress responses?

Cellular stress significantly impacts mitochondrial function and potentially POLRMT activity. Researchers can implement these methodologies:

• Time-Course Experiments: Use the POLRMT antibody in western blotting or immunofluorescence at defined intervals after stress induction (oxidative stress, hypoxia, nutrient deprivation) to track expression changes.

• Live-Cell Imaging: For dynamic studies, complement fixed-cell antibody detection with live-cell imaging using fluorescently tagged POLRMT constructs.

• Subcellular Fractionation: Monitor potential stress-induced changes in POLRMT localization through careful subcellular fractionation followed by immunoblotting.

• Correlation with Mitochondrial Parameters: Simultaneously measure mitochondrial membrane potential, ROS production, and ATP levels alongside POLRMT detection to establish functional correlations during stress responses.

• Analysis of Post-Translational Modifications: Investigate potential stress-induced modifications of POLRMT using phospho-specific antibodies or mass spectrometry following immunoprecipitation.

These approaches provide comprehensive insights into how POLRMT responds to and potentially mediates cellular adaptation to various stressors.

How can POLRMT Antibody, HRP conjugated be incorporated into high-throughput screening platforms?

Adapting POLRMT detection to high-throughput formats requires specific methodological considerations:

• ELISA-Based Screening: The HRP-conjugated POLRMT antibody is particularly suitable for developing ELISA-based screens to identify compounds affecting POLRMT expression or activity. This can be formatted for 96- or 384-well throughput.

• Automated Western Blotting: Simple Western (Wes) or similar automated protein analysis platforms can incorporate the antibody for higher throughput analysis of POLRMT levels across multiple experimental conditions.

• Cell-Based Assays: Develop cell-based assays that correlate POLRMT activity with reporter outputs (e.g., luciferase reporters driven by mitochondrial activity) for functional screens.

• Image-Based High-Content Screening: Optimize immunofluorescence protocols using the primary antibody (non-HRP conjugated) for automated imaging and analysis in high-content screening platforms.

• Multiplexed Detection Systems: Incorporate POLRMT detection into multiplexed assays that simultaneously measure multiple parameters (e.g., mitochondrial function, cell viability) for more informative screening outcomes.

These approaches facilitate the identification of compounds or genetic factors that modulate POLRMT expression or function in a systematic manner.

What are common technical challenges when working with POLRMT Antibody, HRP conjugated and how can they be addressed?

Researchers may encounter several challenges when using POLRMT Antibody, HRP conjugated:

• Non-specific Binding: As documented by manufacturers, some POLRMT antibodies detect a non-specific, cross-reacting band that migrates above POLRMT in western blots . To address this:

  • Include appropriate negative controls (POLRMT knockout/knockdown samples)

  • Optimize antibody dilution to minimize background

  • Consider using alternative blocking reagents (5% BSA vs. milk)

• Variable Signal Intensity: When signal strength is inconsistent:

  • Verify antibody storage conditions

  • Test multiple lots if available

  • Standardize protein extraction protocols

  • Include loading controls for normalization

• Epitope Masking: If POLRMT detection is hindered by protein-protein interactions:

  • Evaluate alternative lysis buffers

  • Consider mild denaturation steps

  • Test different antigen retrieval methods for tissue samples

• HRP Activity Loss: If enzymatic activity diminishes:

  • Minimize freeze-thaw cycles

  • Store in single-use aliquots

  • Protect from light and oxidizing agents

  • Consider adding stabilizing proteins (BSA) to diluted antibody

These troubleshooting approaches can significantly improve experimental outcomes when working with POLRMT antibodies.

How can researchers optimize POLRMT Antibody, HRP conjugated protocols for different sample types?

Protocol optimization should be tailored to specific sample types:

For Cell Lines:

• Lysis Buffer Selection: Compare RIPA, NP-40, and Triton X-100-based buffers to determine optimal POLRMT extraction.

• Protein Loading: Establish standard curves to determine the linear detection range for POLRMT in your system.

• Blocking Optimization: Test 5% milk, 5% BSA, and commercial blocking reagents to minimize background.

For Tissue Samples:

• Homogenization Method: Compare mechanical, enzymatic, and bead-based homogenization for optimal POLRMT preservation.

• Fixation Impact: For fixed tissues, evaluate how different fixatives affect antibody performance.

• Section Thickness: For immunohistochemistry, optimize section thickness (typically 4-7 μm) for best signal-to-noise ratio.

For Primary Cells:

• Gentle Lysis Methods: Primary cells may require gentler lysis conditions to preserve POLRMT integrity.

• Cell Number Optimization: Determine minimum cell numbers required for reliable POLRMT detection.

For All Sample Types:

• Dilution Series: Perform antibody dilution series (1:500 to 1:5000) to identify optimal concentration.

• Incubation Conditions: Test various temperatures (4°C, room temperature) and durations (1 hour to overnight).

• Signal Development: Compare different HRP substrates (ECL, ECL Plus, SuperSignal) for optimal sensitivity and dynamic range.

These optimization strategies ensure robust POLRMT detection across diverse experimental systems.

What approaches should researchers use to quantitatively analyze POLRMT expression data?

Rigorous quantitative analysis of POLRMT expression requires specific methodological considerations:

• Western Blot Densitometry:

  • Use imaging systems with a documented linear dynamic range

  • Include standard curves with recombinant POLRMT protein for absolute quantification

  • Normalize to appropriate loading controls (GAPDH commonly used)

  • Report relative expression changes with proper statistical analysis

• Immunohistochemistry Quantification:

  • Employ digital image analysis with validated algorithms

  • Use H-score or Allred scoring systems for semi-quantitative assessment

  • Implement machine learning approaches for tissue heterogeneity analysis

  • Include positive and negative controls on each slide

• ELISA Data Analysis:

  • Generate standard curves using purified POLRMT protein

  • Ensure samples fall within the linear range of detection

  • Report intra- and inter-assay coefficients of variation

  • Account for matrix effects in complex samples

• Multi-Parameter Correlation:

  • Correlate POLRMT levels with functional outcomes (cell proliferation, mitochondrial function)

  • Apply appropriate statistical methods for multivariate analysis

  • Consider principal component analysis for complex datasets

These quantitative approaches facilitate robust interpretation of POLRMT expression data in various experimental contexts.

How should researchers interpret changes in POLRMT expression in relation to mitochondrial function and disease phenotypes?

Interpreting POLRMT expression changes requires contextual understanding:

• Baseline Expression Comparison: Establish normal POLRMT expression ranges in relevant cell/tissue types. Research has shown significantly higher POLRMT expression in cancer cells compared to normal counterparts .

• Functional Correlation Analysis:

  • Correlate POLRMT levels with mitochondrial transcript levels (NDUFB8, UQCRC2, COXI)

  • Assess relationship with mitochondrial membrane potential

  • Evaluate impact on ROS production and ATP generation

  • Measure effects on cell proliferation and apoptosis

• Disease Context Interpretation:

  • In cancer contexts, elevated POLRMT correlates with increased cell proliferation and migration

  • POLRMT depletion induces apoptosis specifically in cancer cells but not in normal cells

  • POLRMT inhibition affects mitochondrial function through multiple mechanisms

• Mechanistic Framework Development:

  • Integrate POLRMT expression data with broader mitochondrial pathways

  • Consider compensatory mechanisms that may offset POLRMT changes

  • Evaluate time-dependent effects of POLRMT modulation