PREP Antibody

What is PREP and why is it important for research?

PREP (Prolyl Endopeptidase/Prolyl Oligopeptidase) is a serine peptidase that specifically cleaves Pro-Xaa bonds of oligopeptide substrates. It plays a crucial role in hydrolyzing biologically active peptides such as bradykinin, substance P, neurotensin, and vasopressin . PREP's involvement in neuropeptide processing makes it significant for research into cognitive functions, memory processes, and neurological conditions . Research has shown PREP may be linked to conditions including Alzheimer's disease, schizophrenia, and inflammatory disorders, highlighting its value as a potential biomarker and therapeutic target .

What types of PREP antibodies are available for research applications?

Currently, researchers have access to several types of PREP antibodies:

These antibodies have been validated for detecting PREP in various sample types, including cell lysates, serum, plasma, and other biological fluids .

How can researchers optimize PREP antibody use in Western blotting?

For optimal Western blot results with PREP antibodies:

• Sample preparation: Lysates should be prepared using buffer systems that maintain PREP's native structure. Studies have used both sonication and ultracentrifugation (100,000 × g) to separate soluble and membrane fractions .

• Antibody dilution: Optimal dilution varies by antibody; for example, ab58988 has been validated at 1/1000 dilution .

• Predicted band size: PREP typically appears at approximately 81 kDa .

• Controls: Include both positive controls (tissues known to express PREP, such as brain tissue) and negative controls (tissues with PREP knockout or inhibition).

• Optimization: When working with new tissue types, a dilution series is recommended, as PREP expression varies significantly between tissues and cell types .

What are the methodological considerations for using PREP antibodies in ELISA assays?

When using PREP antibodies in ELISA:

• Sandwich ELISA approach: Most commercial PREP ELISA kits use double antibody sandwich methodology to ensure specificity .

• Detection range: Typical assays have a range of 0.156-10 ng/ml with sensitivity below 0.094 ng/ml .

• Sample types: PREP antibody ELISA works with serum, plasma, and cell culture supernatants. Recovery rates vary by matrix type and must be validated for each new application .

• Cross-reactivity: Verify that the antibody doesn't cross-react with other prolyl peptidases such as DPP4, DPP7, DPP8, DPP9, and FAP, which share structural similarities with PREP .

• Storage conditions: Most antibodies maintain stability for 6 months at 4°C under proper storage conditions .

How can researchers use PREP antibodies to investigate PREP's role in peptide processing in the CNS?

Advanced peptidomics approaches incorporating PREP antibodies have revealed significant insights:

• Immunoprecipitation combined with MS analysis: Use PREP antibodies for immunoprecipitation followed by mass spectrometry to identify PREP-associated peptides in neural tissues. This approach has identified both known substrates (substance P, thymosin-β4) and novel substrates .

• Comparative peptidomic analysis: Compare peptidome changes as a function of PREP activity using label-free peptidomics platforms. This methodology revealed that PREP regulates numerous peptides beyond previously known substrates .

• Biochemical validation: After identifying potential substrates through peptidomics, confirm direct PREP-substrate interactions using purified components:

  • Express recombinant wild-type PREP and catalytically inactive mutants (e.g., PrepD35A-K196A)

  • Purify using Ni(II)-affinity chromatography and Superdex 200 columns

  • Conduct in vitro cleavage assays with synthetic peptide substrates

  • Quantify product formation using LC-MS-TOF spectrometry

How do researchers address specificity concerns with PREP antibodies?

Ensuring PREP antibody specificity requires:

• Validation across expression systems: Test antibodies in various cellular expression systems. Studies have used Rosetta2(DE3)pLysS cells for recombinant expression and COS-7 cells for mammalian expression .

• Knockout/knockdown controls: Include PREP-null samples as negative controls.

• Cross-reactivity testing: Test against related prolyl peptidases including PrepL, DPP4, DPP7, DPP8, DPP9 and FAP, which share structural similarities with PREP .

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm binding specificity.

• Isoform specificity: Ensure the antibody recognizes the specific PREP isoform of interest (human PREP spans Leu2-Pro710, accession # P48147) .

What approaches can resolve contradictory results in PREP antibody-based studies?

When facing contradictory results:

• Antibody epitope mapping: Different antibodies may recognize different domains of PREP, potentially yielding varying results depending on protein conformation or post-translational modifications.

• Species-specific variations: Human and mouse PREP show differences that could affect antibody recognition. For example, studies of alpha-synuclein interaction with PREP showed species-specific differences .

• Substrate competition analysis: Some larger peptides like CGRP(1–37) can act as competitive inhibitors rather than substrates, potentially confounding results. Kinetic experiments have shown that CGRP(1–37) can inhibit PREP activity toward smaller substrates like substance P .

• Enzymatic assay correlation: Correlate antibody detection with enzymatic activity assays to ensure the detected PREP is functionally active.

How can PREP antibodies be utilized to study associations between PREP and neurodegenerative disorders?

PREP antibodies enable several key research approaches:

• Comparative tissue analysis: Using Western blotting with PREP antibodies to compare PREP levels in normal vs. pathological brain tissue samples.

• Co-localization studies: Immunohistochemistry with PREP antibodies alongside markers for pathological proteins (e.g., alpha-synuclein, amyloid-beta).

• Protein-protein interaction studies: Research has demonstrated PREP enhances alpha-synuclein dimerization via direct protein-protein interaction, which can be studied using co-immunoprecipitation with PREP antibodies .

• Therapeutic intervention assessment: PREP antibodies can help evaluate the effectiveness of PREP inhibitors, which are being developed as potential treatments for neurodegenerative disorders.

What methodologies exist for using PREP antibodies to study the PI3K/AKT signaling pathway?

Research has identified PREP's role in the PI3K/AKT pathway:

• Signaling pathway analysis: PREP antibodies have been instrumental in demonstrating that PREP regulates IRS-1 stability, which is critical for rapamycin-induced feedback activation of PI3K and AKT .

• Phosphorylation status correlation: Combine PREP antibodies with phospho-specific antibodies to examine correlations between PREP levels and phosphorylation states of pathway components.

• Inhibitor studies: Use PREP antibodies to monitor changes in PREP expression/localization following treatment with pathway inhibitors.

• Co-immunoprecipitation: Use PREP antibodies to identify novel protein interactions within the PI3K/AKT pathway.

How can researchers use PREP antibodies to investigate PREP's role in cancer biology?

PREP antibodies have helped uncover connections between PREP and cancer:

• Tumor tissue analysis: Compare PREP expression in normal vs. tumor tissues using immunohistochemistry or Western blotting with PREP antibodies.

• Combined targeting studies: Research has examined combined targeting of Fibroblast Activation Protein and PREP using pseudopeptide inhibitors, with PREP antibodies serving as critical tools for mechanism validation .

• Functional studies: Use PREP antibodies to monitor changes in PREP expression/activity following treatment with potential anti-cancer compounds.

• Mechanistic insights: Investigate PREP's interaction with cancer-relevant signaling pathways using PREP antibodies for immunoprecipitation followed by proteomic analysis.

What are the latest methodological advances in using PREP antibodies for high-throughput screening applications?

Recent advances include:

• Multiplexed assay systems: Integration of PREP antibody detection with other biomarkers in multiplex platforms.

• Automated image analysis: Machine learning algorithms that can analyze PREP immunostaining patterns across large tissue sample sets.

• Activity-based protein profiling: Combining PREP-specific activity-based probes with PREP antibodies for functional proteomics.

• High-content screening: Using PREP antibodies in cell-based high-content screening to identify compounds that modulate PREP expression, localization, or activity.

How do broadly neutralizing antibodies (bNAbs) differ from traditional PrEP approaches?

While this represents a different use of the term "PrEP", researchers should understand:

What methodological considerations exist for research involving antibody-based HIV prevention?

When researching antibody-based PrEP:

• Viral sensitivity testing: Determine the neutralization profile of target virus populations using panels of different viruses from various geographic regions .

• Antibody production approaches: New methods like anti-idiotypic monoclonal antibodies (AI-mABs) can stimulate B-cells to produce specific bNAbs in larger amounts with greater purity .

• Combination strategies: Current research focuses on cocktails of multiple bNAbs rather than single antibodies to neutralize a broader range of viral isolates .

• Pharmacokinetic considerations: Research has shown that concomitant use of traditional PrEP (tenofovir-emtricitabine) with antibody-based approaches may affect antibody clearance rates, potentially reducing efficacy .

• Biological markers: Studies have noted associations between antiretroviral PrEP use, increased intestinal permeability (measured by I-FABP levels), and enhanced antibody clearance .

How might next-generation sequencing technologies enhance PREP antibody research?

Emerging opportunities include:

• Single-cell approaches: Combining PREP antibody-based cell sorting with single-cell RNA sequencing to characterize PREP-expressing cell populations.

• Spatial transcriptomics: Correlating PREP protein localization (detected by antibodies) with spatial gene expression patterns.

• Epitope mapping: Using next-generation sequencing to map the precise binding sites of various PREP antibodies on the PREP protein.

• Antibody engineering: Computational approaches to design improved PREP antibodies with enhanced specificity and sensitivity.

What are the key methodological questions for researchers developing therapeutic applications targeting PREP?

Critical considerations include:

• Specificity challenges: How to ensure therapeutic approaches specifically target PREP without affecting related prolyl peptidases.

• Target validation: How PREP antibodies can be used to validate PREP as a therapeutic target across different disease models.

• Biomarker development: Methods to use PREP antibodies for developing companion diagnostics for PREP-targeted therapeutics.

• Pharmacodynamic markers: Approaches to use PREP antibodies to monitor target engagement and biological effects of PREP inhibitors in clinical samples.