PPID Antibody

What is PPID and why are antibodies against it important in research?

PPID (Peptidylprolyl Isomerase D) is a member of the cyclophilin-type PPIase family that accelerates protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . Antibodies against PPID are crucial research tools for:

• Studying protein folding mechanisms

• Investigating the role of PPID in disease models

• Examining protein-protein interactions involving PPID

• Understanding PPID's function in cellular pathways

The inactivation of the PPID gene has been shown to rescue the disease phenotype of Col6a1 deficiency, highlighting its potential role in certain pathological conditions .

What applications are PPID antibodies commonly used for?

PPID antibodies have been validated for multiple research applications:

Multiple studies have successfully employed these applications to investigate PPID's biological functions and interactions .

What is the molecular weight of PPID and how does this impact antibody selection?

PPID has a calculated molecular weight of 41 kDa (370 amino acids), but is typically observed at 38-41 kDa in SDS-PAGE . When selecting an antibody:

• Verify that the antibody detects the appropriate molecular weight band

• Be aware that PPID antibodies sometimes detect a weaker band at approximately 70 kDa due to crosslinking

• Consider that post-translational modifications may affect apparent molecular weight

• Choose antibodies validated for your specific application and species of interest

Understanding the expected molecular weight helps distinguish specific signals from non-specific binding and ensures accurate interpretation of experimental results.

How should I design kinetic analysis experiments for PPID protein-protein interactions?

When designing kinetic analysis experiments for PPID protein-protein interactions:

• Critical experimental controls: Include both positive and negative controls to validate interactions. Data analysis alone is insufficient to discriminate between different reaction schemes .

• Duration considerations: Vary injection durations when using surface plasmon resonance (SPR) to distinguish between different binding models .

• Recovery analysis: Collect and reanalyze fractions from antibody surfaces to confirm binding specificity .

• Surface considerations: When immobilizing antibodies, test surfaces with and without dextran matrix—binding curves should be superimposable if the interaction is specific .

• Apply CF-PPiD technology: Consider using cell-free protein array technology for proximity biotinylation-based PPI identification, which has been shown to effectively detect PPID interactions .

Remember that experimental design, not just data analysis, is key to successful interaction analysis .

What cross-reactivity concerns should I address when using PPID antibodies?

When working with PPID antibodies, several cross-reactivity considerations must be addressed:

• Verify antibody specificity: Some PPID antibodies are specifically engineered to show "no cross-reactivity with other proteins" , but this should always be independently validated.

• Species cross-reactivity: Check if the antibody cross-reacts across species. Many PPID antibodies react with human, mouse, and rat PPID , but specificity can vary.

• Potential cross-reaction with SecY: Some α-PpiD antibodies have been observed to cross-react with purified SecY in some experimental setups, though this cross-reactivity may only occur with purified protein and not in membrane preparations .

• Validation in knockout models: To definitively confirm antibody specificity, test in PPID knockout samples. Cross-linking products observed with PPID antibodies should be absent in ΔppiD strains .

• Controls for cross-reactivity: Include isotype controls and pre-absorption controls using the immunizing peptide when possible.

To minimize cross-reactivity issues, select antibodies targeting unique epitopes of PPID and validate specificity in your experimental system.

How can PPID antibodies be used to study the dynamic interaction between PPID and the Sec translocon?

PPID (PpiD in bacteria) has been identified as a component of the Sec translocon complex through several advanced techniques:

• UV cross-linking with pBpa incorporation: Using SecY with site-specific incorporation of p-benzoyl-L-phenylalanine (pBpa) at position I91, researchers demonstrated that PPID binds to the lateral gate of SecY. This interaction creates a UV-specific cross-linking product of approximately 105 kDa that is recognized by α-PpiD antibodies .

• Blue native PAGE analysis: This technique revealed two forms of PPID in E. coli membranes:

  • A 70 kDa form representing monomeric PPID or PPID-YfgM complex

  • A 300 kDa complex containing PPID-SecYEG

• Confirmation in knockout strains: The 300 kDa band was absent in ΔppiD strains, confirming specificity .

• Functional studies: Research has shown that PPID is detached from the SecY complex by nascent membrane proteins but not by SecA, suggesting a dynamic regulatory role .

To study these interactions, combine immunoprecipitation with PPID antibodies, blue native PAGE separation, and mass spectrometry analysis to identify interacting partners under different conditions.

What techniques can be employed to investigate PPID's role in protein folding pathways?

To investigate PPID's role in protein folding pathways, several sophisticated techniques can be employed:

• Proximity-based biotinylation:

  • Use CF-PPiD technology (Cell-free protein array for proximity biotinylation-based PPI identification) to detect direct interaction partners of PPID

  • This method can identify both strong and weak interactions that might be missed by traditional co-immunoprecipitation

• Cis-trans isomerase activity assays:

  • Design assays using specific peptide substrates containing proline residues

  • Monitor conformational changes using spectroscopic techniques (fluorescence, circular dichroism)

  • Compare wild-type PPID activity with that of mutated versions

• Gene inactivation studies:

  • Examine phenotypic changes in PPID knockout models

  • Assess rescue effects in disease models (e.g., Col6a1 deficiency models)

• Structural biology approaches:

  • Use PPID antibodies for co-crystallization studies

  • Employ cryo-EM to visualize PPID in complex with client proteins

• Time-resolved techniques:

  • Apply pulse-chase experiments with PPID antibodies to track dynamic interactions

  • Use FRET-based assays to monitor real-time interactions in living cells

These approaches provide complementary information about PPID's catalytic mechanism and its interaction network in protein folding pathways.

How can I optimize Western blot protocols for PPID detection?

Optimizing Western blot protocols for PPID detection requires attention to several key factors:

• Sample preparation:

  • For brain tissue samples, use homogenization in RIPA buffer with protease inhibitors

  • For cell lines (A549, Jurkat, MCF-7, NIH/3T3), lyse cells in buffer containing 1% NP-40 or similar detergents

• Protein loading and separation:

  • Load 20-30 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal separation around the 38-41 kDa range

• Antibody selection and dilution:

  • For polyclonal antibodies like #12716-1-AP, use dilutions of 1:500-1:2000

  • Commercial antibodies typically range from 0.2-1 mg/ml concentration

• Detection bands and specificity:

  • Expect primary band at 38-41 kDa

  • Be aware of potential weak band at ~70 kDa due to crosslinking

  • To verify specificity, use PPID knockout samples as negative controls

• Positive controls:

  • Use mouse brain tissue, A549 cells, Jurkat cells, or MCF-7 cells as positive controls

  • K562 cell lysates have been validated for Cyclophilin 40 expression

Following these optimizations should result in clear, specific detection of PPID in Western blot applications.

What are the key considerations when selecting between polyclonal and monoclonal PPID antibodies?

When choosing between polyclonal and monoclonal PPID antibodies, consider these research-critical factors:

For PPID research specifically, consider that:

• Polyclonal antibodies like HPA019520 or 12716-1-AP are well-validated for multiple applications

• Monoclonal antibodies like clone 4C7 may offer more consistent results for longitudinal studies

• Your experimental goals should dictate the choice between broader epitope recognition (polyclonal) versus highly specific detection (monoclonal)

How can PPID antibodies be employed in studying post-translational modifications of PPID?

Studying post-translational modifications (PTMs) of PPID using antibodies requires specialized approaches:

• PTM-specific antibodies:

  • While the search results don't specifically mention PPID PTM-specific antibodies, this approach would involve developing antibodies that specifically recognize phosphorylated, acetylated, or otherwise modified PPID

  • Validation would require comparing signals between treated and untreated samples

• Immunoprecipitation followed by PTM detection:

  • Use PPID antibodies for immunoprecipitation (IP) at recommended dilutions (0.5-4.0 μg for 1-3 mg total protein)

  • Follow with Western blotting using PTM-specific antibodies (anti-phospho, anti-acetyl, etc.)

  • This approach allows enrichment of PPID before PTM analysis

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Use PPID antibodies for Western blotting to detect charge shifts indicating PTMs

  • Compare patterns under different cellular conditions

• Mass spectrometry integration:

  • Immunoprecipitate PPID using validated antibodies

  • Analyze by mass spectrometry to identify PTM sites

  • Quantify PTM changes under different experimental conditions

• Proximity labeling approaches:

  • Adapt CF-PPiD technology to study how PTMs affect PPID's interaction network

  • Compare biotinylation patterns between wild-type and mutant PPID (with modified PTM sites)

These techniques provide complementary information about how PTMs regulate PPID function and interactions in different cellular contexts.

How can PPID antibodies be integrated into proximity biotinylation techniques for protein interaction studies?

PPID antibodies can be strategically integrated with proximity biotinylation techniques for comprehensive protein interaction studies:

• CF-PPiD technology implementation:

  • The recently developed Cell-free Protein array for Proximity biotinylation-based PPI identification (CF-PPiD) offers a high-throughput approach for PPID interaction studies

  • This method combines human protein arrays (containing 19,712 recombinant proteins) with proximity biotinylation enzymes like AirID

  • PPID antibodies can validate interactions identified through this screening approach

• Validation workflow:

  • Use CF-PPiD to identify potential PPID interaction partners

  • Confirm these interactions using co-immunoprecipitation with PPID antibodies

  • For weak interactions that may not be detected by traditional IP, use proximity biotinylation followed by streptavidin pulldown

• Combining approaches for weak interactions:

  • Some proteins (like ZBTB9 in IκBα studies) may be highly biotinylated in proximity assays but not detected by immunoprecipitation

  • PPID antibodies can be used to validate these weak/transient interactions through alternative methods

• Drug-inducible interaction studies:

  • Similar to studies with AirID-fused cereblon that showed drug-inducible PPIs using CF-PPiD

  • PPID antibodies can confirm these conditional interactions under various treatments

This integrated approach leverages the specificity of PPID antibodies with the sensitivity of proximity biotinylation to comprehensively map PPID's protein interaction network.

What role might PPID play in disease mechanisms based on recent research findings?

Recent research suggests several potential roles for PPID in disease mechanisms that merit further investigation using PPID antibodies:

• Collagen VI-related myopathies:

  • PPID gene inactivation has been shown to rescue the disease phenotype of Col6a1 deficiency

  • This suggests PPID may function as a disease modifier in certain muscular disorders

  • PPID antibodies can be used to study expression levels and localization in patient samples

• Protein folding disorders:

  • As a peptidyl-prolyl isomerase, PPID catalyzes a rate-limiting step in protein folding

  • Dysregulation could contribute to protein misfolding diseases

  • Antibodies can track PPID expression and localization changes in disease models

• SecY translocon interactions:

  • PPID has been identified as a component of the SecYEG translocon complex

  • This interaction suggests a role in membrane protein biogenesis

  • Disruption of this interaction could potentially impact cellular proteostasis

• Potential therapeutic target:

  • The finding that PPID inactivation can rescue certain disease phenotypes suggests it might be a therapeutic target

  • PPID antibodies are crucial tools for validating target engagement in drug development

• Biomarker potential:

  • Changes in PPID expression or localization could serve as biomarkers for certain diseases

  • Immunohistochemistry using PPID antibodies on tissue microarrays could identify such patterns

These emerging research directions highlight the importance of well-characterized PPID antibodies in advancing our understanding of disease mechanisms and potentially developing new therapeutic approaches.