PPIF Antibody

What is PPIF and why is it important in research?

PPIF (Peptidylprolyl isomerase F), also known as cyclophilin F, CYP3, PPIase F, or Rotamase F, belongs to the cyclophilin-type PPIase family . It functions as a mitochondrial matrix protein that catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, accelerating protein folding . PPIF is a key component of the mitochondrial permeability transition pore (MPTP) in the inner mitochondrial membrane and plays crucial roles in regulating apoptotic and necrotic cell death pathways . This makes PPIF a significant target for studies in mitochondrial biology, cell death mechanisms, and diseases associated with mitochondrial dysfunction such as lung adenocarcinoma (LUAD) .

What applications are PPIF antibodies validated for?

According to the available data, PPIF antibodies have been validated for multiple experimental applications:

It is recommended to optimize antibody concentration for each specific experimental system to obtain optimal results .

How should I store and handle PPIF antibodies to maintain activity?

PPIF antibodies should be stored at -20°C and remain stable for one year after shipment . For PBS-only formulations, storage at -80°C is recommended . The standard storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, but some preparations (20μl sizes) may contain 0.1% BSA . When handling the antibody, avoid repeated freeze-thaw cycles and exposure to direct light to maintain optimal antibody activity and specificity.

What controls should I include when using PPIF antibodies?

For rigorous experimental design with PPIF antibodies, include the following controls:

• Positive control: Use tissues or cell lines known to express PPIF, such as HeLa cells, HepG2 cells, or heart tissue (human, mouse, or rat) .

• Negative control: Include samples where PPIF is known to be absent or samples from PPIF knockout models.

• Primary antibody omission control: Process samples without the primary antibody to evaluate background staining.

• Isotype control: Use a non-specific antibody of the same isotype (e.g., Rabbit IgG for polyclonal or Mouse IgG2b for monoclonal antibodies) to assess non-specific binding .

• Loading control: For Western blots, include housekeeping proteins (e.g., GAPDH, β-actin) to normalize expression levels.

How do I optimize antigen retrieval for PPIF IHC in different tissue types?

• Perform a time-course experiment (10, 20, 30 minutes) for antigen retrieval to determine optimal duration.

• Compare heat-induced epitope retrieval methods (microwave, pressure cooker, water bath) for your specific tissue.

• For tissues with high lipid content, add 0.1% Tween-20 to retrieval buffers to enhance penetration.

• For formalin-fixed tissues with extensive cross-linking, extend retrieval time and consider enzymatic retrieval (proteinase K) as a supplementary approach.

• Validate optimization with positive control tissues (human heart) alongside your experimental tissues.

Remember that overly harsh antigen retrieval can damage tissue morphology, while insufficient retrieval may result in false-negative staining.

What are the key considerations when analyzing PPIF expression in mitochondrial studies?

When investigating PPIF in mitochondrial research contexts, several methodological considerations are critical:

• Subcellular localization confirmation: As PPIF is a mitochondrial matrix protein, co-localization studies with established mitochondrial markers (e.g., TOM20, MitoTracker) are essential to confirm proper localization .

• Mitochondrial isolation protocols: Standard cell lysis buffers may not effectively extract mitochondrial proteins. Use specialized mitochondrial isolation buffers containing sucrose and mannitol to maintain mitochondrial integrity.

• Functional assays: Since PPIF regulates the mitochondrial permeability transition pore (MPTP), complement expression analysis with functional assessments such as calcium retention capacity assays or mitochondrial swelling assays.

• Oxidative modifications: PPIF function can be altered by post-translational modifications during oxidative stress. Consider redox proteomics approaches when studying PPIF in pathological conditions.

• Dynamic regulation: PPIF may translocate between submitochondrial compartments in response to stress. Time-course analyses with subcellular fractionation can reveal these dynamics.

How can I address high background or non-specific binding issues with PPIF antibodies?

Persistent background or non-specific binding when working with PPIF antibodies can significantly impact data interpretation. Implement these systematic troubleshooting strategies:

• Blocking optimization: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) to determine which most effectively reduces background in your specific application.

• Antibody cross-reactivity: PPIF belongs to the cyclophilin family with structural similarities to other members. The 18466-1-AP antibody has been immunized with full-length PPIF and absorbed by PPIA to enhance specificity . If cross-reactivity persists, consider:

  • Performing peptide competition assays with recombinant PPIF

  • Using molecular weight verification (expected at 22 kDa)

  • Increasing washing stringency with higher salt concentrations or detergent

• Tissue-specific autofluorescence: For IF/ICC applications, particularly in tissues with high autofluorescence (e.g., brain, liver), incorporate additional steps:

  • Pre-treatment with Sudan Black B

  • Photobleaching before antibody incubation

  • Using fluorophores that emit in ranges distinct from tissue autofluorescence

• Secondary antibody optimization: Test secondary antibodies from different suppliers and consider highly cross-adsorbed secondary antibodies to minimize species cross-reactivity.

How should I design experiments to study PPIF's role in the mitochondrial permeability transition pore?

Investigating PPIF's functional role in the MPTP requires a multi-faceted experimental approach:

• Genetic manipulation strategies: Implement PPIF knockdown (siRNA, shRNA) or knockout (CRISPR-Cas9) approaches alongside overexpression models. When using these models, verify changes in PPIF expression using the validated antibody dilutions (WB: 1:1000-1:4000) .

• Pharmacological interventions: Incorporate PPIF inhibitors (e.g., Cyclosporin A) alongside genetic approaches to distinguish between acute and chronic effects of PPIF modulation.

• MPTP functional assays: Combine PPIF expression analysis with functional readouts:

  • Calcium retention capacity assays

  • Mitochondrial swelling measurements

  • TMRM-based membrane potential assessments

  • Cytochrome c release quantification

• Interaction studies: Use PPIF antibodies in co-immunoprecipitation experiments to identify PPIF-interacting proteins within the MPTP complex. The recommended amount is 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Stress-response dynamics: Design experiments that monitor PPIF localization and expression following specific stress stimuli (oxidative stress, calcium overload, ischemia-reperfusion) using immunofluorescence at 1:10-1:100 dilution .

What methodological approaches can resolve contradictory PPIF expression data in different experimental systems?

Resolving contradictory PPIF expression data across experimental systems requires systematic investigation of technical and biological variables:

• Antibody epitope considerations: Different antibodies recognize distinct epitopes that may be differentially accessible in various experimental conditions. The PAT1F5AT monoclonal antibody targets human Cyclophilin-F amino acids 3-27 , while polyclonal antibodies may recognize multiple epitopes. Map all available antibodies to specific PPIF domains and compare results.

• Post-translational modifications: PPIF function is regulated by PTMs including phosphorylation and oxidation. Employ:

  • Phospho-specific antibodies

  • Redox proteomics approaches

  • 2D gel electrophoresis to resolve modified PPIF variants

• Isoform-specific expression: Verify whether contradictory results stem from detection of different PPIF isoforms. Design isoform-specific primers for RT-qPCR validation alongside Western blot analysis.

• Technical standardization:

  • Implement absolute quantification using recombinant PPIF standards

  • Standardize sample preparation protocols, particularly for mitochondrial isolation

  • Use multiple detection methods (WB, IF, IHC) in parallel

• Biological variables documentation: Systematically document cell confluence, passage number, tissue origin, and physiological state, as these factors can significantly influence mitochondrial protein expression patterns.

How can I effectively use PPIF antibodies in multiplexing experiments with other mitochondrial markers?

Multiplexing experiments with PPIF antibodies and other mitochondrial markers require careful planning to avoid signal overlap and cross-reactivity:

• Antibody selection strategy:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-PPIF with mouse anti-TOM20)

  • Ensure antibodies have been validated in multiplexing applications

  • For same-species antibodies, use directly conjugated primary antibodies or sequential staining protocols with intermediate blocking steps

• Spectral considerations for fluorescent detection:

  • Select fluorophores with minimal spectral overlap

  • Include single-stain controls for spectral unmixing

  • Consider fluorescence lifetime imaging microscopy (FLIM) to distinguish overlapping signals

• Optimization for specific mitochondrial structures:

  • For co-localization with outer membrane proteins: Use mild permeabilization (0.1% Triton X-100)

  • For matrix protein co-localization: Use stronger permeabilization (0.5% Triton X-100)

  • Adjust fixation protocols to preserve mitochondrial morphology (4% PFA with 0.1% glutaraldehyde)

• Quantitative co-localization analysis:

  • Implement Pearson's correlation coefficient, Manders' overlap coefficient, and intensity correlation analysis

  • Use super-resolution techniques (STED, STORM) for precise localization

• Controls for multiplexed experiments:

  • Include single primary antibody controls with all secondary antibodies to detect cross-reactivity

  • Use cells with known PPIF expression patterns (HeLa, HepG2, MCF-7) as staining optimization controls

What methodological approaches are recommended for studying PPIF as a prognostic marker in cancer research?

Based on recent findings linking PPIF expression to prognosis in lung adenocarcinoma (LUAD) , implementing rigorous methodological approaches for cancer prognostic studies requires:

How can I investigate the role of PPIF in modulating immune cell infiltration in cancer?

Recent research has implicated PPIF in immune regulation, particularly in balancing T helper 1-T helper 2 cell responses in LUAD . To investigate these immunomodulatory functions:

• Multiplex immunohistochemistry approach:

  • Develop multiplexed panels including PPIF (1:100-1:400) and lineage-specific immune cell markers

  • Implement spectral imaging and automated quantification algorithms

  • Analyze spatial relationships between PPIF-expressing cells and immune infiltrates

• Flow cytometry methodology:

  • Optimize PPIF detection in conjunction with immune cell surface markers

  • Design panels to identify specific T cell subsets (Th1, Th2, Treg)

  • Include intracellular cytokine staining to correlate with functional state

• In vitro immune cell co-culture systems:

  • Establish co-culture systems with PPIF-manipulated tumor cells and immune cells

  • Measure cytokine profiles using multiplex assays

  • Assess functional immune readouts (proliferation, cytotoxicity)

• Single-cell analysis techniques:

  • Apply single-cell RNA-seq to tumor samples with known PPIF status

  • Perform CyTOF (mass cytometry) to simultaneously measure multiple immune parameters

  • Use trajectory analysis to identify potential differentiation pathways affected by PPIF

• Validation in mouse models:

  • Compare immune infiltration in PPIF-overexpressing vs. PPIF-knockout tumor models

  • Use adoptive transfer experiments to track specific immune populations

  • Implement immune checkpoint blockade to assess therapeutic implications

What experimental design is optimal for investigating PPIF's role in mitophagy through the FOXO3a/PINK1-Parkin pathway?

Recent studies indicate that PPIF can impede mitophagy by targeting the FOXO3a/PINK1-Parkin signaling pathway . An optimal experimental design to investigate this mechanism includes:

• Genetic manipulation systems:

  • Create stable cell lines with inducible PPIF expression

  • Implement CRISPR-Cas9-based PPIF knockout models

  • Use site-directed mutagenesis to generate PPIF variants with altered binding capabilities

• Real-time mitophagy monitoring:

  • Employ mt-Keima or mito-QC reporter systems for pH-based mitophagy detection

  • Use live-cell imaging with PPIF-fluorescent protein fusions (verify function is maintained)

  • Quantify mitochondrial mass using MitoTracker and flow cytometry

• Molecular pathway analysis:

  • Assess FOXO3a phosphorylation status using phospho-specific antibodies

  • Monitor PINK1 stabilization on the outer mitochondrial membrane

  • Quantify Parkin recruitment to mitochondria via fractionation and immunoblotting

  • Measure ubiquitination of mitochondrial outer membrane proteins

• Mitochondrial stress induction protocols:

  • Implement standardized mitophagy triggers (CCCP, antimycin A, hypoxia)

  • Develop time-course experiments to capture dynamic processes

  • Include positive controls (PINK1/Parkin overexpression)

• Biochemical interaction studies:

  • Perform co-immunoprecipitation with PPIF antibodies to identify binding partners

  • Use proximity ligation assays to verify protein-protein interactions in situ

  • Conduct in vitro binding assays with recombinant proteins to determine direct interactions

What are the critical parameters for quantitative PPIF detection by ELISA?

For accurate quantification of PPIF in research samples using ELISA:

• Sample preparation optimization:

  • For plasma and serum samples: Use protease inhibitor cocktails during collection

  • For cell culture samples: Ensure complete cell lysis with mitochondria-specific extraction buffers

  • Consider subcellular fractionation to enrich for mitochondrial proteins

• Assay standardization:

  • Create standard curves using recombinant PPIF proteins

  • Validate linearity across the anticipated concentration range

  • Include quality control samples with known PPIF concentrations

• Technical validation parameters:

  • Determine lower and upper limits of quantification

  • Establish intra- and inter-assay coefficients of variation

  • Verify specificity through spike-and-recovery experiments

  • Test for matrix effects with dilution linearity studies

• Analytical considerations:

  • Use four-parameter logistic regression for standard curve fitting

  • Implement blank subtraction to account for background signal

  • Normalize to total protein concentration when appropriate

• Control recommendations:

  • Include samples from tissues known to express high levels of PPIF (heart tissue)

  • Run parallel Western blot analysis for cross-method validation

Available ELISA kits employ a sandwich enzyme immunoassay technique with polyclonal antibodies specific for human PPIF pre-coated onto 96-well microplates, enabling quantitative measurement in approximately 4 hours .

How should I design experiments to investigate PPIF post-translational modifications?

Since PPIF function is regulated by post-translational modifications, comprehensive characterization requires:

• Phosphorylation analysis strategy:

  • Use phospho-specific antibodies when available

  • Implement phospho-enrichment techniques (IMAC, TiO2) prior to mass spectrometry

  • Conduct in vitro kinase assays to identify responsible kinases

  • Apply lambda phosphatase treatment as a control to verify phosphorylation-specific bands

• Oxidative modification detection:

  • Use redox proteomics approaches (BIAM labeling, dimedone-based probes)

  • Implement differential alkylation strategies to identify reversible oxidation

  • Conduct site-directed mutagenesis of redox-sensitive residues

  • Use reducing/oxidizing agents to confirm reversibility of modifications

• Ubiquitination and SUMOylation analysis:

  • Perform immunoprecipitation under denaturing conditions

  • Use deubiquitinase inhibitors during sample preparation

  • Implement tandem ubiquitin-binding entities (TUBEs) for enrichment

  • Conduct mass spectrometry with specific fragmentation methods for ubiquitin remnants

• Mass spectrometry workflow optimization:

  • Consider top-down proteomics to maintain intact modification patterns

  • Implement multiple proteolytic enzymes to maximize sequence coverage

  • Use electron-transfer dissociation for labile modifications

  • Develop targeted methods for known modification sites

• Functional correlation studies:

  • Correlate PTM patterns with PPIF activity in the MPTP

  • Generate PTM-mimetic mutations to assess functional consequences

  • Study PTM dynamics under different cellular stress conditions

What are the emerging applications for PPIF antibodies in translational research?

Based on current research trends, several promising translational applications for PPIF antibodies are emerging:

• Biomarker development: PPIF's prognostic value in LUAD suggests potential as a clinical biomarker . Standardized IHC protocols with validated antibody dilutions (1:100-1:400) could be implemented in clinical pathology workflows.

• Therapeutic target validation: As PPIF regulates the MPTP, which is implicated in ischemia-reperfusion injury and neurodegenerative diseases, antibodies are essential tools for validating drug target engagement in preclinical studies.

• Personalized medicine approaches: PPIF expression patterns could potentially guide therapeutic decisions, particularly for treatments targeting mitochondrial function or cell death pathways.

• Immune response modulation: Given PPIF's role in T helper cell balance , antibody-based monitoring of PPIF in immune contexts could inform immunotherapy approaches.

• Combination therapy development: PPIF antibodies can help elucidate mechanisms of synergy between mitochondria-targeting compounds and established therapeutics.

Future technological developments, including automated digital pathology and multiplexed imaging techniques, will likely expand the utility of PPIF antibodies in clinical research settings.

What methodological gaps remain in PPIF antibody applications and how might they be addressed?

Despite significant progress in PPIF antibody development and application, several methodological challenges persist:

• Isoform-specific detection: Current antibodies may not distinguish between potential PPIF splice variants or post-translationally modified forms. Development of isoform-specific antibodies through careful epitope selection could address this limitation.

• Dynamic monitoring limitations: Conventional antibody applications provide static snapshots rather than dynamic information. Integration with emerging technologies such as optogenetics or FRET-based biosensors could provide real-time insights into PPIF function.

• Cross-species reactivity inconsistencies: While some antibodies show reactivity with human, mouse, and rat samples , comprehensive cross-species validation is lacking. Systematic epitope mapping and conservation analysis could improve cross-species applications.

• Subcellular resolution challenges: Standard microscopy may be insufficient to precisely localize PPIF within mitochondrial subcompartments. Super-resolution microscopy and proximity labeling techniques could enhance spatial resolution.

• Quantification standardization: Absolute quantification methods for PPIF across different experimental systems remain unstandardized. Development of calibrated reference materials and digital PCR-based absolute quantification could improve cross-study comparability.