POLR1D Antibody

What applications are validated for POLR1D antibodies?

POLR1D antibodies have been validated for multiple experimental applications, with varying degrees of optimization for each technique. Successful applications include Western blotting (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation (IP) .

When designing experiments, consider the following application-specific dilution recommendations:

Note that these dilutions should be optimized for your specific experimental system, as optimal conditions can vary depending on sample type, preparation method, and detection system .

What cell and tissue types have been validated with POLR1D antibodies?

Validation studies have confirmed successful detection of POLR1D in multiple cell lines and tissue types. For research design purposes, consider using the following positively validated samples for controls:

When performing IHC with POLR1D antibodies, optimal results may be achieved using TE buffer pH 9.0 for antigen retrieval, though citrate buffer pH 6.0 can serve as an alternative .

How should POLR1D antibody storage and handling be optimized for experimental reproducibility?

For maximum stability and consistent results, POLR1D antibodies should be stored at -20°C and remain stable for approximately one year after shipment when properly handled . The following practices are recommended to maintain antibody integrity:

• Store in aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce antibody activity

• When provided in glycerol solutions (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3), aliquoting may be unnecessary for -20°C storage

• Smaller volume products (e.g., 20μl sizes) often contain 0.1% BSA as a stabilizer

• When thawing, allow the antibody to reach room temperature completely before use

• Avoid contamination by using sterile technique when handling

Failing to follow these storage recommendations can lead to reduced sensitivity and inconsistent results across experiments .

How can POLR1D antibodies be utilized to investigate cancer progression mechanisms?

Recent research has implicated POLR1D in cancer progression, making it a valuable target for oncological studies. When designing experiments to investigate POLR1D's role in cancer:

What are the key considerations when using POLR1D antibodies for developmental biology research?

POLR1D plays critical roles in embryonic development, with mutations linked to developmental disorders such as Treacher Collins syndrome . When designing developmental biology experiments:

• Consider the timing of POLR1D expression analysis, as research indicates:

  • POLR1D function is essential for early embryonic development

  • POLR1D mutant embryos cannot be recovered at E7.5 early post-gastrulation stage, suggesting failed implantation

  • Complete loss of POLR1D results in embryonic lethality prior to blastocyst stage

• Focus on ribosomal RNA (rRNA) synthesis assessment, as POLR1D is required for rRNA production:

  • Use RT-qPCR to measure rRNA levels in wild-type versus POLR1D mutant/knockdown models

  • A Drosophila line with the POLR1D G30R mutation (orthologous to the human TCS-causing G52E mutation) showed reduced rRNA levels

• Incorporate apoptosis and reactive oxygen species (ROS) detection:

  • TUNEL labeling to detect apoptotic cells in POLR1D-deficient developmental models

  • CellROX® Green Reagent (5 μM) for ROS detection, incubating samples at 37°C for 30 minutes

How can discrepancies in observed POLR1D molecular weight between theoretical prediction and experimental results be addressed?

Researchers often observe discrepancies between the calculated molecular weight of POLR1D (15-16 kDa) and its apparent molecular weight in experimental conditions (20 kDa) . When troubleshooting these differences:

• Consider post-translational modifications as a primary cause of mobility shifts:

  • Phosphorylation, glycosylation, or ubiquitination can increase apparent molecular weight

  • Run parallel samples with phosphatase treatment to determine if phosphorylation contributes to the shift

• Validate antibody specificity using multiple approaches:

  • Compare results with different POLR1D antibody clones (e.g., 16678-1-AP and 12254-1-AP)

  • Perform siRNA knockdown experiments to confirm band identity

  • Include positive control lysates from validated cell lines (Jurkat, HeLa, HepG2)

• Optimize electrophoresis conditions:

  • Use 5-20% gradient SDS-PAGE gels for better resolution of lower molecular weight proteins

  • Run gels at 70V (stacking)/90V (resolving) for 2-3 hours for optimal separation

  • Include molecular weight markers that cover the 10-25 kDa range

What is the optimal Western blot protocol for POLR1D detection?

For reproducible and specific detection of POLR1D by Western blotting, follow this optimized protocol based on validated research approaches:

• Sample preparation:

  • Load 30 μg of protein per lane under reducing conditions

  • Validated sample types include HeLa, Jurkat, HepG2, and HEL whole cell lysates, as well as mouse and rat lung tissue lysates

• Electrophoresis parameters:

  • Use 5-20% SDS-PAGE gel

  • Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

• Transfer conditions:

  • Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with POLR1D antibody at 0.5-1.0 μg/mL overnight at 4°C

  • Wash with TBS-0.1% Tween 3 times, 5 minutes each

  • Probe with appropriate anti-rabbit IgG-HRP secondary antibody at 1:5000 dilution for 1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) detection system

  • Expect to observe a specific band for POLR1D at approximately 20 kDa (though the theoretical molecular weight is 15-16 kDa)

How can POLR1D antibodies be utilized to investigate its role in ribosomal RNA synthesis?

To study POLR1D's function in ribosomal RNA synthesis, consider implementing these methodological approaches:

• Quantitative assessment of rRNA levels:

  • Extract total RNA using a high-quality isolation kit

  • Synthesize cDNA using standard protocols (e.g., iScript cDNA synthesis kit)

  • Perform RT-qPCR with primers spanning exon junctions to prevent genomic DNA amplification

  • For POLR1D: 5′-ACGATCAGGAGCTGGAGAGA and 5′-TGCTGGCAGACATTCAAGAG

  • For housekeeping control (Actb): 5'-GGCCCAGAGCAAGAGAGGTATCC and 5'-ACGCACGATTTCCCTCTCAGC

• Functional POLR1D knockdown:

  • Design multiple siRNAs targeting POLR1D (at least two different sequences)

  • Transfect into appropriate cell lines (e.g., SK-MES-1, H2170)

  • Confirm knockdown efficiency by Western blot

  • Measure rRNA levels using RT-qPCR in control vs. knockdown cells

• Analysis of POLR1D interaction with POLR1C:

  • Perform co-immunoprecipitation using POLR1D antibodies

  • Investigate how mutations in POLR1D (such as G52E in humans or G30R/E in Drosophila) affect heterodimerization with POLR1C in vitro

  • Consider examining the impact of these interactions on rRNA synthesis and cell proliferation

What are the key considerations for using POLR1D antibodies in cancer prognosis research?

When employing POLR1D antibodies for cancer prognosis studies, consider these methodological approaches based on successful research outcomes:

How can POLR1D antibodies be used to investigate potential therapeutic targets for cancer treatment?

Recent research suggests POLR1D may be a potential therapeutic target for cancer treatment, particularly through its interaction with the PI3K-Akt pathway . Consider these methodological approaches for investigating POLR1D as a therapeutic target:

• Mechanism-driven therapeutic target validation:

  • Combine POLR1D knockdown with PI3K/Akt pathway inhibitors to assess synergistic effects

  • Measure changes in proliferation, migration, and invasion using CCK-8 and transwell assays

  • Quantify effects on downstream signaling molecules by Western blotting

• In vivo model development:

  • Establish xenograft models using cells with POLR1D knockdown or overexpression

  • Monitor tumor growth, metastasis, and response to treatment

  • Evaluate POLR1D expression in patient-derived xenografts to assess clinical relevance

• Drug sensitivity profiling:

  • Screen cancer cell lines with varying POLR1D expression levels for sensitivity to chemotherapeutic agents

  • Investigate whether POLR1D expression levels predict response to specific treatments

  • Focus on ribosome biogenesis inhibitors, as POLR1D functions in rRNA synthesis

What are the key considerations when using POLR1D antibodies for developmental disorder research?

POLR1D mutations have been linked to Treacher Collins syndrome (TCS), a condition affecting facial development . For developmental disorder research:

• Mutation-specific analysis:

  • Focus on specific mutations such as the G52E in human POLR1D or orthologous mutations in model organisms (e.g., G30R in Drosophila)

  • Use immunofluorescence to assess POLR1D localization in cells with TCS-causing mutations

  • Combine with POLR1C antibodies to investigate effects on heterodimer formation

• Neural crest cell (NCC) investigation:

  • Examine POLR1D expression and function in neural crest cells, as TCS results from restrictions on NCC migration

  • Use lineage tracing combined with POLR1D antibody staining in developmental models

  • Assess p53 activation in POLR1D-deficient neural progenitors, as POLR1D loss-of-function increases p53 expression

• Ribosome biogenesis assessment:

  • Measure nucleolar stress and ribosome biogenesis defects in POLR1D mutant models

  • Investigate the relationship between ribosome biogenesis and p53-dependent apoptosis in neural tissues

  • Consider combining with markers of nucleolar stress to assess cellular responses to POLR1D dysfunction

How can discrepancies in experimental results across different POLR1D antibodies be reconciled?

When confronted with conflicting results from different POLR1D antibodies, consider these methodological approaches to reconcile discrepancies:

• Epitope mapping and antibody characterization:

  • Determine the specific epitopes recognized by different antibody clones

  • Consider whether antibodies target regions affected by known splice variants or post-translational modifications

  • Assess whether discrepancies might result from differential recognition of modified forms of POLR1D

• Multi-antibody validation strategy:

  • Use at least two different antibodies targeting distinct epitopes of POLR1D

  • Compare results from monoclonal and polyclonal antibodies

  • Implement genetic approaches (siRNA, CRISPR) to validate antibody specificity

• Cell and tissue-specific expression analysis:

  • Evaluate whether discrepancies stem from cell-type specific post-translational modifications

  • Compare antibody performance across different cell lines and tissue types

  • Consider species-specific differences when working with human, mouse, and rat samples

  • Note that while POLR1D antibodies show reactivity with human, mouse, and rat samples, specific epitope recognition may vary