PPRC1 Antibody

What is PPRC1 and why is it important in scientific research?

PPRC1 (Peroxisome Proliferator-Activated Receptor Gamma, Coactivator-Related 1) is the third member of the PGC1 family that contributes to mitochondrial biogenesis and orchestrates responses to metabolic stress. It promotes the expression of multiple genes related to inflammation, proliferation, and metabolic reprogramming . Recent research has identified PPRC1 as a potential biomarker in various cancers, particularly ovarian and hepatocellular carcinoma, where its expression correlates with prognosis, immune cell infiltration, and tumor-stemness indices .

What applications are PPRC1 antibodies validated for?

PPRC1 antibodies have been validated for multiple applications:

It is recommended to optimize antibody concentration for each specific application and sample type to obtain optimal results .

What species reactivity can I expect from commercially available PPRC1 antibodies?

Most commercially available PPRC1 antibodies demonstrate reactivity with:

When selecting an antibody for your experiment, verify the specific reactivity profile provided by the manufacturer, as reactivity can vary between different antibody products .

How should I optimize Western blot protocols for PPRC1 detection?

For optimal Western blot detection of PPRC1:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors

• Protein loading: Load 20-50 μg of total protein per lane

• Electrophoresis conditions: Use 8-10% gels as PPRC1 has a high molecular weight (observed at approximately 177 kDa)

• Transfer conditions: Employ overnight transfer at low voltage for high molecular weight proteins

• Blocking: Use 5% BSA in TBS-T for 1 hour at room temperature

• Primary antibody incubation: Dilute antibody 1:200-1:1000 in 1% BSA in TBS-T and incubate overnight at 4°C

• Validation controls: Include positive controls (NIH/3T3 cells, C6 cells) where PPRC1 expression has been confirmed

What is the recommended protocol for immunohistochemical detection of PPRC1?

For immunohistochemical detection:

• Tissue preparation: Use freshly prepared 10% formalin-fixed, paraffin-embedded sections (4-6 μm)

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ and non-specific binding with 5% normal serum

• Primary antibody: Apply diluted antibody and incubate overnight at 4°C

• Detection system: Use appropriate HRP-conjugated secondary antibody and DAB substrate

• Counterstaining: Counterstain with hematoxylin

• Controls: Include positive tissue controls (heart, skeletal muscle) and negative controls (omitting primary antibody)

This protocol may require optimization based on specific tissue type and fixation conditions .

How can PPRC1 antibodies be used to study its role in cancer progression?

PPRC1 antibodies can be employed in multiple advanced applications to investigate its role in cancer:

What are the methodological considerations when examining PPRC1 in relation to immune checkpoint expression?

When investigating relationships between PPRC1 and immune checkpoints:

• Multiplex immunofluorescence:

  • Use PPRC1 antibody in conjunction with antibodies against immune checkpoint molecules (PD-1, PD-L1, CTLA-4)

  • Employ spectral unmixing to resolve signal overlap

  • Analyze co-expression at single-cell resolution

• Flow cytometry:

  • Combine surface staining for immune checkpoints with intracellular PPRC1 staining

  • Use appropriate permeabilization protocols optimized for nuclear proteins

• Correlation analysis approaches:

  • For tissue analyses, use consecutive sections for PPRC1 and immune checkpoint staining

  • Perform Pearson or Spearman correlation analyses between PPRC1 expression and immune checkpoint genes

  • Consider multivariate analyses to account for confounding factors

Research has revealed significant positive correlations between PPRC1 expression and 47 immune checkpoint genes in multiple cancer types, suggesting PPRC1 may influence immunotherapy responses .

How can I validate the specificity of PPRC1 antibody signals in my experiments?

To ensure antibody specificity:

• Positive and negative controls:

  • Use cell lines with known PPRC1 expression (NIH/3T3, C6 cells are positive controls)

  • Include tissues known to express PPRC1 (heart, skeletal muscle show high expression; brain shows moderate expression)

• Knockdown/knockout validation:

  • Perform siRNA knockdown of PPRC1 (similar to methods used in PGC-1α knockdown studies)

  • Compare antibody signal between wildtype and knockdown samples

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of PPRC1

  • Compare staining patterns for consistency

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

What are the common pitfalls in interpreting PPRC1 expression data?

Researchers should be aware of several potential issues:

• Isoform specificity: Up to two different isoforms have been reported for PPRC1 . Verify which isoform(s) your antibody detects.

• Molecular weight variations: While the calculated molecular weight of PPRC1 is 165 kDa, the observed molecular weight is approximately 177 kDa . This discrepancy may lead to misidentification of bands.

• Post-translational modifications: Consider that phosphorylation or other modifications may alter antibody binding or protein migration patterns.

• Cross-reactivity: Despite manufacturer claims of specificity, validate the absence of cross-reactivity with other PGC family members (PGC-1α, PGC-1β).

• Subcellular localization: PPRC1 is primarily nuclear ; cytoplasmic staining may indicate non-specific binding or altered biology.

• Threshold determination: When categorizing samples as "high" versus "low" expression, use appropriate statistical methods (e.g., median split as used in cancer prognosis studies) .

How can I overcome weak or absent PPRC1 signal in Western blot analyses?

If experiencing detection issues with PPRC1 in Western blot:

• Protein extraction optimization:

  • Use nuclear extraction protocols as PPRC1 is primarily nuclear

  • Add phosphatase inhibitors to preserve potential phosphorylated forms

• Antibody concentration:

  • Increase antibody concentration within recommended range (1:200-1:1000)

  • Extend primary antibody incubation time to overnight at 4°C

• Sample preparation:

  • Avoid repeated freeze-thaw cycles of protein samples

  • Use freshly prepared samples when possible

• Detection system:

  • Switch to more sensitive detection systems (e.g., chemiluminescent substrates with enhanced sensitivity)

  • Consider signal amplification methods for low-abundance targets

• Transfer conditions:

  • For high molecular weight proteins like PPRC1 (177 kDa), use:

    • Lower percentage gels (8%)

    • Longer transfer times (overnight)

    • Addition of SDS (0.1%) to transfer buffer

What methods should I use to quantify PPRC1 levels in patient samples for prognostic studies?

For quantitative assessment of PPRC1 in clinical samples:

• Immunohistochemistry scoring:

  • Use validated scoring systems like H-score (combining intensity and percentage of positive cells)

  • Employ digital pathology with AI-assisted quantification for reproducibility

  • Have multiple independent pathologists score to reduce subjectivity

• ELISA-based quantification:

  • Use sandwich ELISA kits with detection range of 0.156-10 ng/mL

  • Follow standardized protocols for sample collection and processing

  • Include standard curves for accurate quantification

• RNA-based methods:

  • Combine with protein detection for comprehensive analysis

  • Use RT-qPCR with validated reference genes

  • Consider RNA-seq for broader transcriptomic context

• Normalization strategies:

  • For Western blot: normalize to appropriate loading controls

  • For IHC: use tissue microarrays with control tissues

  • For ELISA: use validated reference standards

• Statistical analysis:

  • Use appropriate statistical methods to establish cutoff values

  • Consider both continuous and categorical analyses

  • Perform multivariate analysis to account for confounding variables

How can I distinguish between PPRC1 and other PGC family members in my research?

To differentiate PPRC1 from other PGC family members:

• Antibody selection:

  • Choose antibodies raised against unique epitopes not conserved in PGC-1α or PGC-1β

  • Verify specificity through testing in models with known expression patterns

• Expression pattern analysis:

  • PPRC1 is highly expressed in heart and skeletal muscle, moderately in lung, placenta, intestine, liver, kidney, spleen, thymus, colon, and brain

  • Compare with known tissue-specific expression of other family members

• Functional studies approach:

  • Unlike PGC-1α, PPRC1 expression does not directly correlate with expression of TFAM and SDHA in some contexts

  • Design experiments to distinguish specific downstream targets

• Knockout/knockdown validation:

  • Perform selective knockdown of PPRC1 versus other family members

  • Analyze differential effects on mitochondrial biogenesis and target gene expression

• Co-expression analysis:

  • Examine correlation between PPRC1 and other family members

  • Research shows PPRC1 and PGC-1α may have cooperative rather than redundant roles in mitochondrial function

What are the differences in research methodologies when studying PPRC1 versus PGC-1α in mitochondrial function?

When investigating mitochondrial function:

• Target gene selection:

  • For PPRC1: Focus on TFAM and SDHA, which show good correlation with PPRC1 expression levels

  • For PGC-1α: Examine broader set of mitochondrial genes that may not correlate with PPRC1

• Experimental design considerations:

  • PPRC1 responds more rapidly to serum stimulation than PGC-1α

  • Design time-course experiments to capture these temporal differences

• Stress response studies:

  • PPRC1 orchestrates responses to metabolic stress differently than PGC-1α

  • Test multiple stress conditions (nutrient deprivation, oxidative stress) to differentiate responses

• Knockdown effects:

  • PGC-1α knockdown leads to slight decreases in TFAM and MT-CO2

  • PPRC1 knockdown may have distinct mitochondrial phenotypes

• Cancer context analysis:

  • PPRC1 has stronger correlations with cancer prognosis in certain contexts

  • Design studies to compare predictive value of both proteins in the same cancer types

How can PPRC1 antibodies be utilized to explore its roles in emerging research areas?

PPRC1 antibodies can facilitate investigation of several frontier research areas:

• Cancer immunotherapy biomarkers:

  • Use PPRC1 antibodies to stratify patients for immunotherapy response

  • Investigate correlation between PPRC1 expression and immune checkpoint inhibitor efficacy

  • Research shows PPRC1 expression significantly correlates with immune checkpoint genes

• Mitochondrial dynamics in disease:

  • Employ super-resolution microscopy with PPRC1 antibodies to track localization during mitochondrial stress

  • Combine with metabolic flux analysis to link PPRC1 levels with metabolic reprogramming

• Drug development targeting PPRC1 pathway:

  • Screen compounds that modify PPRC1 expression or function

  • Use antibodies to monitor target engagement in drug screens

  • Example: Eugenol has been shown to downregulate PPRC1 and induce apoptosis in leukemia cells

• Mutation-specific antibodies:

  • Develop antibodies targeting common PPRC1 mutations (e.g., P941T frameshift mutation found in multiple cancers)

  • Use these to study functional consequences of specific mutations

• Multi-omic integration:

  • Combine antibody-based proteomics with genomics and transcriptomics

  • Correlate PPRC1 protein levels with mutation status and transcriptional networks

What methodological approaches should be considered when investigating PPRC1 mutations in cancer?

For researchers investigating PPRC1 mutations:

• Mutation hotspot analysis:

  • Focus on proline residues at positions 938, 940, and 941, which are frequently mutated across 11 cancer types

  • Design primers or antibodies specific to these mutation sites

• Functional characterization methods:

  • Use CRISPR-Cas9 to introduce specific mutations (e.g., P941Tfs frameshift)

  • Assess consequences on protein stability, localization, and function

  • Compare wildtype and mutant PPRC1 interactomes through IP-MS

• Clinical correlation approaches:

  • Develop mutation-specific detection methods for patient stratification

  • Correlate mutation status with:

    • Treatment response

    • Survival outcomes

    • Immune infiltration profiles

• Comprehensive mutation screening:

  • Consider the range of mutation types (frameshift, missense, nonsense) observed in PPRC1

  • Table of common mutations found across cancer types: