PPIA Antibody Pair

What is PPIA and why are antibody pairs against this target significant in research?

PPIA (Peptidylprolyl Isomerase A/Cyclophilin A) is an 18 kDa protein that belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) family. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerates protein folding .

PPIA antibody pairs are significant because they enable:

• Detection of PPIA in multiple experimental contexts (Western blotting, immunohistochemistry, flow cytometry)

• Quantification of PPIA expression levels through sandwich ELISA techniques

• Investigation of PPIA's roles in HIV-1 replication, cancer progression, and protein translation

• Study of PPIA interactions with other proteins like NRF2 in cancer pathways

The canonical protein structure has 165 amino acid residues and can be found in the nucleus, cytoplasm, and extracellular space .

What are the key considerations when selecting antibody pairs for PPIA detection?

When selecting antibody pairs for PPIA detection, researchers should consider:

• Epitope targeting: Ensure antibodies recognize distinct, non-overlapping epitopes. From the literature, antibodies targeting the C-terminal region (like ABIN2855908) and those targeting N-terminal regions can form effective pairs .

• Host species diversity: Select antibodies from different host species (e.g., rabbit and mouse) to avoid cross-reactivity in sandwich assays.

• Validation status: Prioritize antibodies validated in multiple applications with knockout validation where possible .

• Species cross-reactivity: Consider whether the antibodies need to work across species (human, mouse, rat) based on your experimental model .

• Performance in specific applications: Some antibodies perform better in certain applications than others. For instance, the Picoband antibody A01308 shows strong performance in Western blot, IHC, ICC, and flow cytometry .

How can I map and validate the specific epitopes recognized by my PPIA antibody pair?

Epitope mapping is crucial for ensuring your antibody pair targets distinct epitopes. Based on the comparative study of epitope mapping technologies (search result 4), we recommend:

• Hydrogen-Deuterium Exchange (HDX): This technique identified epitopes in 5/5 antibody-antigen pairs tested and provides high-resolution mapping of conformational epitopes .

• Chemical Cross-Linking (XL): While it missed some antibody CDR3 residues, XL provides clear interaction interfaces between antibody and antigen, with identified residues typically 15-30Å apart .

• Alanine Scanning Mutagenesis: This approach can identify critical residues for antibody binding.

• Peptide Array Analysis: While this approach worked for only 2/5 antibody-antigen pairs in the study, it may be useful for linear epitopes in PPIA .

For PPIA specifically, antibodies targeting the C-terminal region (residues 116-165) have been documented for successful detection .

What experimental controls are essential when using PPIA antibody pairs in co-immunoprecipitation studies?

When conducting co-immunoprecipitation studies with PPIA antibody pairs:

• Negative controls:

  • IgG isotype control to assess non-specific binding

  • Lysates from PPIA-knockout cells or tissues (when available)

  • Competitive blocking with recombinant PPIA protein

• Positive controls:

  • Known PPIA interaction partners (e.g., NRF2 as demonstrated in search result 3)

  • Recombinant PPIA-tagged protein

• Validation approaches:

  • Reciprocal co-IP (pull down with anti-PPIA and probe for interactor, then reverse)

  • Use of catalytically inactive PPIA mutants (e.g., PPIA R55A&F60A) to distinguish enzyme activity-dependent interactions

  • Treatment with cyclosporin A (CsA) which blocks PPIA interactions, as demonstrated with NRF2

As shown in the study of PPIA-NRF2 interactions, co-immunoprecipitation revealed that PPIA's enzymatic activity is required for the interaction, since no binding was observed with catalytically dead PPIA variants .

How can PPIA antibody pairs be optimized for studying PPIA's role in HIV-1 infection?

To study PPIA's role in HIV-1 infection:

• Selecting appropriate antibodies: Choose antibodies that don't interfere with the Gag-binding region of PPIA, as this interaction is critical for HIV-1 replication .

• Critical experimental design elements:

  • Include proper controls with cyclosporin A (CsA), which disrupts PPIA-Gag interactions

  • Consider genetic approaches using cells from individuals with different PPIA SNPs

  • Examine both virion-incorporated and cellular PPIA separately

• Genetic considerations: The SNP3 and SNP4 promoter polymorphisms in PPIA are associated with more rapid CD4+ T-cell loss and disease progression in HIV-infected individuals . Design experiments to compare PPIA levels in cells with different genotypes using your antibody pairs.

According to research, "Cyclophilin A binds to the Gag protein of human immunodeficiency virus type 1 (HIV-1)" and "may have an essential function in HIV-1 replication" .

What methodological approaches are recommended for using PPIA antibody pairs to investigate protein translation mechanisms?

Recent research has revealed PPIA's role in supporting translation of intrinsically disordered proteins . When investigating this function:

• Experimental design considerations:

  • Use paired antibodies to detect both PPIA and its substrate proteins (~20% of which engage in protein phase separation)

  • Employ PPIA inhibitors (e.g., TMN355) as controls to confirm translation effects are PPIA-dependent

  • Consider combined approaches (e.g., IP-western blot) to validate proteomic findings

• Translation assay approaches:

  • Fluorescent aminoacyl-tRNA analogue staining can detect de novo translation rates in stem cells with/without PPIA inhibition

  • Combine with RNA sequencing to distinguish transcriptional vs. translational effects

• Control methodologies:

  • Use Ppia knockout and heterozygous models

  • Monitor expression of IDR-rich PPIA substrates in both epithelial and hematopoietic cell lines

Research demonstrates "a significantly decreased rate of de novo translation in stem cells that had been treated with the potent PPIA inhibitor TMN355," confirming PPIA's direct role in protein translation .

What special considerations apply when using PPIA antibody pairs to investigate cancer progression mechanisms?

PPIA plays important roles in cancer progression, particularly through NRF2 stability in lung cancer . When investigating these mechanisms:

• Experimental approach:

  • Use antibody pairs that can detect both PPIA and its cancer-relevant partners (e.g., NRF2)

  • Design co-immunoprecipitation experiments to capture protein-protein interactions

  • Include controls with catalytically inactive PPIA variants (PPIA R55A&F60A)

• Validation strategies:

  • siRNA knockdown of PPIA to confirm antibody specificity

  • Ubiquitination assays to confirm PPIA's effect on protein stability

  • Pull-down analysis using recombinant PPIA-conjugated beads

• Technical considerations:

  • CsA treatment blocks PPIA-NRF2 interaction and can serve as an important control

  • PPIA's enzymatic activity is required for interaction with NRF2, so consider this when selecting antibodies

Research shows that "si PPIA treatment profoundly elevated the NRF2 ubiquitination" and that "PPIA's enzymatic activity is required for the PPIA-NRF2 interaction" .

What are the most common technical challenges when working with PPIA antibody pairs in flow cytometry, and how can they be overcome?

Common challenges in flow cytometry with PPIA antibodies include:

• Intracellular accessibility issues:

  • Solution: Ensure proper fixation (4% paraformaldehyde) and permeabilization as demonstrated in the Boster Bio protocols

  • Validation: Compare to isotype controls and unlabeled samples

• Non-specific binding:

  • Solution: Block with 10% normal goat serum before antibody incubation

  • Validation: Include rabbit IgG isotype controls at equivalent concentrations

• Signal optimization:

  • Recommended antibody concentration: 1μg/1×10^6 cells

  • Incubation conditions: 30 min at 20°C

  • Secondary antibody: DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1×10^6 cells)

The validated protocol from search result 6 demonstrates successful detection of PPIA in THP-1, U937, and K562 cells with distinct positive population separation from controls .

How should disagreements in PPIA detection between different antibody-based methodologies be interpreted and resolved?

When facing discrepancies between different antibody-based methodologies:

• Common sources of discrepancy:

  • Epitope accessibility differences between applications

  • Post-translational modifications affecting antibody recognition

  • Protein conformation differences in native vs. denatured conditions

• Systematic resolution approach:

  • Validation with multiple antibodies: Use antibodies targeting different PPIA epitopes

  • Complementary techniques: Combine antibody-based detection with mass spectrometry

  • Genetic validation: Use PPIA knockout controls or siRNA knockdown

• Application-specific considerations:

  • Western blotting may detect denatured epitopes not accessible in native conditions

  • IHC/ICC may show different results due to fixation effects on epitope accessibility

  • ELISA sensitivity often differs from Western blot

The search results show that some antibodies display different molecular weights than expected (e.g., 185 kDa observed vs. 18 kDa calculated) , which may indicate detection of complexes, dimers, or post-translationally modified forms.

What optimization strategies are recommended for developing a sandwich ELISA for PPIA quantification?

For developing a sandwich ELISA for PPIA:

• Antibody pair selection:

  • Choose antibodies targeting non-overlapping epitopes

  • Consider using a monoclonal capture antibody and polyclonal detection antibody

  • Available ELISA kits demonstrate detection ranges from 15.6 pg/mL to 100 μg/mL

• Optimization parameters:

  • Coating concentration: Typically 1-10 μg/mL of capture antibody

  • Sample diluent: Include blocking proteins to prevent non-specific binding

  • Detection sensitivity: Commercial kits report sensitivities ranging from 0.087 ng/mL to 0.78 ng/mL

• Validation approach:

  • Generate standard curves using recombinant PPIA

  • Test recovery with spiked samples

  • Perform parallelism testing with diluted biological samples

Commercial ELISA kits have established detection ranges of "3.12-200ng/ml" with sensitivities as low as "0.087 ng/mL" .

How can PPIA antibody pairs be employed to investigate the role of PPIA in aging and stem cell biology?

Recent research has revealed PPIA's importance in stem cell biology and aging:

• Experimental design considerations:

  • Use antibody pairs to detect PPIA in hematopoietic stem cells

  • Combine with functional assays to correlate PPIA levels with stem cell function

  • Compare young vs. aged stem cell populations

• Key methodological approaches:

  • Immunofluorescence with stem cell markers

  • Flow cytometry to quantify PPIA in specific stem cell populations

  • Co-immunoprecipitation to identify age-dependent PPIA interaction partners

• Validation strategies:

  • Genetic approaches with Ppia heterozygous and knockout models

  • Rescue experiments to demonstrate causal relationships between PPIA and aging phenotypes

Research has shown that "To substantiate the causal relationship between PPIA and the ageing phenotype, we subsequently performed rescue experiments" in hematopoietic stem cells .

What are the considerations for developing multiplexed detection systems that include PPIA antibody pairs?

For multiplexed detection systems incorporating PPIA antibodies:

• Technical requirements:

  • Ensure antibodies are compatible with multiplexing reagents

  • Select antibodies from different host species to avoid cross-reactivity

  • Consider differential labeling strategies (fluorophores, quantum dots)

• Validation approach:

  • Test for signal bleed-through between channels

  • Verify antibody performance in single-plex before multiplexing

  • Include single-stained controls in multiplex experiments

• Application-specific considerations:

  • For flow cytometry: carefully select fluorophores with minimal spectral overlap

  • For multiplexed IHC: sequential staining may be preferable to simultaneous staining

  • For bead-based multiplexing: test for cross-reactivity with other capture antibodies

Multiplexed approaches allow simultaneous detection of PPIA and its interaction partners or downstream effectors, such as NRF2 in cancer studies .

How can genetic variation in PPIA be accounted for when developing and using antibody pairs?

Genetic variation in PPIA can affect antibody recognition and experimental outcomes:

• Key genetic variations to consider:

  • SNP3 and SNP4 in the promoter region affect PPIA expression levels

  • Other polymorphisms may affect protein structure or post-translational modifications

• Experimental design considerations:

  • When possible, genotype samples for known PPIA polymorphisms

  • Include samples with different PPIA genotypes as controls

  • Consider that some epitopes may be affected by genetic variants

• Antibody selection strategy:

  • Choose antibodies targeting conserved regions when working across genetically diverse samples

  • Use multiple antibodies targeting different epitopes

  • Validate antibodies against samples with known PPIA variants

Research has shown that "individuals who processed two functional variants in the promoter region of PPIA had higher risk of CD4+ T-cell loss or progression to AIDS-defining diseases" .