PRCP Antibody, FITC conjugated

What is PRCP and what is its function?

PRCP (prolylcarboxypeptidase, also known as angiotensinase C) is a serine protease that plays critical roles in cellular signaling pathways. Research has demonstrated that PRCP, alongside the related enzyme PREP (prolyl endopeptidase), regulates proliferation and survival in various cancer cells, including breast and pancreatic cancer lines. Mechanistically, PRCP functions as a stabilizer of Insulin Receptor Substrate-1 (IRS-1), which is critical for PI3K/AKT/mTOR signaling pathways . This stabilization helps maintain active signaling through these proliferation and survival pathways, making PRCP a potential therapeutic target in cancer research.

Why is FITC conjugation used for PRCP antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides a direct fluorescent tag that enables visualization and quantification of PRCP in various experimental applications without requiring secondary antibodies. The fluorescent properties of FITC allow researchers to detect PRCP expression using flow cytometry, fluorescence microscopy, and other fluorescence-based techniques . FITC emits green fluorescence when excited with blue light (488 nm), making it compatible with standard laboratory equipment. Furthermore, FITC conjugation facilitates multi-parameter analyses where different targets can be simultaneously detected using antibodies conjugated to spectrally distinct fluorophores, similar to multi-color cocktails used for T-cell population analysis .

What are the recommended dilutions for PRCP-FITC antibody in different applications?

The optimal dilution of PRCP-FITC antibody varies depending on the specific application:

• FLISA (Fluorescence-Linked Immunosorbent Assay): 1:1000 dilution

• Western Blot: 1:100-500 dilution

• Immunochemistry: 1:10-50 dilution

These recommendations serve as starting points, and researchers should perform antibody titration experiments to determine the optimal concentration for their specific experimental system, cell types, and instrumentation. Higher concentrations may be needed for applications requiring detection of low-abundance PRCP, while lower concentrations might be sufficient for systems with high PRCP expression levels. Importantly, the product information emphasizes that the antibody should not be frozen, which could affect its binding capacity and fluorescence properties .

How does PRCP's role in the PI3K/AKT pathway impact experimental design?

Understanding PRCP's role in the PI3K/AKT pathway is crucial for designing experiments that accurately assess its function. PRCP and PREP regulate IRS-1 stability, which directly impacts PI3K activity and subsequent AKT phosphorylation . This relationship should inform several aspects of experimental design:

• Appropriate controls must include assessment of IRS-1 levels and phosphorylation status, particularly at serine residues 307 and 636/639 .

• Experiments should measure downstream pathway components, including PI3K activity and AKT phosphorylation, to fully capture PRCP's functional impact .

• Time-course experiments are essential since PRCP/PREP depletion affects IRS-1 half-life, requiring protein turnover assessment .

• Combination studies with pathway inhibitors (like rapamycin or LY294002) may reveal complex feedback mechanisms, as PRCP/PREP depletion can block rapamycin-induced feedback activation of AKT .

• Cell viability and apoptosis assays should be included to connect molecular changes to functional outcomes, as the PI3K/AKT pathway regulates these processes .

How does PRCP function in cancer cell signaling pathways?

PRCP, alongside PREP, functions as a critical regulator in cancer cell signaling by maintaining IRS-1 stability. Research has established that PRCP/PREP activity prevents inhibitory serine phosphorylation of IRS-1, thereby preserving active PI3K/AKT signaling . This mechanism has several important implications:

• PRCP/PREP depletion or inhibition induces serine phosphorylation of IRS-1 (at Ser-307 and Ser-636/639), triggering IRS-1 degradation and subsequent inactivation of PI3K and AKT .

• In pancreatic cancer cells, depletion of PRCP/PREP significantly decreases PI3K activity (measured by in vitro kinase assays), demonstrating direct regulation of this pathway .

• Reduced PI3K activity and AKT phosphorylation following PRCP/PREP depletion correlates with decreased cell viability and increased apoptosis, highlighting the functional significance of this regulation .

• While PRCP/PREP depletion affects IRS-1 stability and PI3K/AKT activation, mTOR activity (measured by S6K phosphorylation) remains relatively unchanged, indicating selective pathway regulation .

• PRCP/PREP also modulates response to rapamycin (an mTOR inhibitor), as their depletion blocks rapamycin-induced increases in IRS-1 levels and AKT phosphorylation that normally occur through feedback mechanisms .

This detailed understanding positions PRCP as a potential therapeutic target in cancers where the PI3K/AKT pathway drives proliferation and survival.

What mechanisms explain how PRCP stabilizes IRS-1 in signaling pathways?

The precise mechanisms by which PRCP stabilizes IRS-1 involve sophisticated regulation of post-translational modifications. Research findings suggest several potential mechanisms:

• PRCP/PREP activity prevents inhibitory serine phosphorylation of IRS-1, particularly at sites including Ser-307 and Ser-636/639 . When PRCP/PREP are depleted, these phosphorylation events increase, targeting IRS-1 for degradation.

• The regulation appears to involve both mTOR/S6K-sensitive mechanisms (affecting Ser-307 phosphorylation) and mTOR/S6K-insensitive mechanisms (affecting Ser-636/639 phosphorylation) . This suggests PRCP operates through multiple regulatory pathways.

• PRCP/PREP depletion shortens the half-life of IRS-1 protein without affecting IRS-1 mRNA levels, confirming post-translational regulation rather than transcriptional effects .

• In rapamycin-treated cells, PRCP/PREP depletion still causes IRS-1 degradation despite blocking of mTOR/S6K activity, further supporting the existence of mTOR-independent mechanisms .

• PRCP/PREP inhibition with ZPP (Z-Pro-Prolinal) produces effects similar to genetic depletion, suggesting their enzymatic activity is required for IRS-1 stabilization .

These findings indicate that PRCP and PREP likely function as upstream regulators that protect IRS-1 from multiple degradation-inducing phosphorylation events, thereby maintaining PI3K/AKT signaling capacity.

How can PRCP-FITC antibodies be combined with other markers in multiparameter flow cytometry?

Multiparameter flow cytometry using PRCP-FITC antibodies requires careful experimental design to optimize detection while avoiding spectral overlap. When designing such experiments, researchers should consider:

• Fluorophore selection: FITC emits in the green spectrum (~520 nm), so complementary fluorophores should be selected to minimize spectral overlap. Common complementary fluorophores include:

  • PE (phycoerythrin) for red emission (~575 nm)

  • PerCP or PerCP-Cyanine5.5 for far-red emission (~675-710 nm)

  • APC (allophycocyanin) for far-red emission (~660 nm)

• Sample preparation protocol: For optimal multiparameter detection, follow protocols similar to established antibody cocktails:

  • Add appropriate volumes of each antibody to 100 μL of sample

  • Incubate at 4°C in the dark for 30 minutes

  • Process samples according to cell type (e.g., RBC lysis for blood samples)

  • Wash with appropriate buffer and analyze promptly

• Compensation controls: Single-stained controls for each fluorophore are essential for accurate compensation, especially when FITC is combined with PE which has some spectral overlap .

• Gating strategy: Begin with forward/side scatter to identify cells of interest, followed by dead cell exclusion, then analyze PRCP-FITC expression in conjunction with other markers such as CD3, CD4, or CD45 depending on your research question .

• For complex studies combining PRCP-FITC with markers of signaling pathways (like phospho-AKT), consider using markers conjugated to fluorophores that excite with different lasers to minimize compensation requirements.

How does PRCP inhibition synergize with mTOR inhibitors in cancer models?

The synergistic effect between PRCP inhibition and mTOR inhibitors represents a significant finding with therapeutic implications. Research demonstrates this synergy occurs through multiple mechanisms:

• Rapamycin (an mTOR inhibitor) normally causes feedback activation of IRS-1-PI3K-AKT signaling, which can limit its therapeutic efficacy. When PRCP and PREP are depleted or inhibited, this feedback activation is blocked .

• Mechanistically, PRCP/PREP depletion prevents the rapamycin-induced increase in IRS-1 protein levels, thereby preventing PI3K activation and subsequent AKT phosphorylation .

• In vitro kinase assays confirmed that while rapamycin increases IRS-1- and p85-associated PI3K activity in control cells, PRCP/PREP depletion significantly reduces both basal and rapamycin-induced PI3K activation .

• Combination treatment with the PRCP/PREP inhibitor ZPP and rapamycin demonstrated synergistic cytotoxicity (CI<1) in pancreatic cancer cell lines including Panc-1 and PK-9 .

• The synergistic effect was observed across multiple pancreatic cancer cell lines (Panc-1, PK-9, and Capan-1), suggesting broad applicability .

• While rapamycin primarily inhibits mTORC1 and ZPP inhibits PRCP/PREP, their combination effectively blocks both mTOR signaling and the compensatory activation of the PI3K/AKT pathway, resulting in enhanced cancer cell death .

This synergy provides a rational basis for combination therapy approaches targeting both mTOR and PRCP/PREP in cancers where PI3K/AKT signaling drives tumor growth and survival.

What are optimal controls for validating PRCP-FITC antibody specificity?

Rigorous validation of PRCP-FITC antibody specificity requires comprehensive controls that address multiple potential sources of artifacts:

• Epitope-specific controls:

  • Peptide competition: Pre-incubate PRCP-FITC antibody with the N-terminal PRCP peptide (the immunizing epitope) to confirm binding specificity

  • Alternative antibody validation: Compare staining patterns with antibodies targeting different PRCP epitopes

• Expression-based controls:

  • Positive control: Include cell lines known to express PRCP (e.g., pancreatic cancer lines like Panc-1)

  • Negative control: Use cell lines with no/low PRCP expression

  • Genetic validation: Compare staining between wild-type cells and PRCP knockdown/knockout cells

• Technical controls:

  • Isotype control: Use rabbit IgG-FITC at matching concentration to assess non-specific binding

  • Unstained control: Measure cellular autofluorescence

  • Secondary-only control: For indirect detection methods in comparative studies

  • Fluorescence-minus-one (FMO) controls: Essential for accurate gating in multiparameter flow cytometry

• Functional validation:

  • Correlate PRCP-FITC staining with functional readouts like IRS-1 stability or PI3K/AKT activation

  • Verify that cells with stronger PRCP-FITC signal show expected phenotypes (e.g., increased IRS-1 stability)

  • Demonstrate that PRCP-FITC signal decreases with expected kinetics following PRCP inhibition

These controls ensure that experimental findings truly reflect PRCP biology rather than technical artifacts or non-specific signals.

How should samples be prepared for optimal PRCP antibody staining?

Sample preparation significantly impacts PRCP antibody staining quality and specificity. The following methodological approaches optimize results across different applications:

• Flow cytometry preparation:

  • For cell suspensions: Harvest cells using enzyme-free methods if possible to preserve surface epitopes

  • For blood samples: Use 5 μL antibody per 100 μL anticoagulant-treated blood

  • Incubate at 4°C in the dark for 30 minutes

  • For blood samples, use appropriate RBC lysis solution

  • Wash with cell staining buffer containing 2-5% protein (BSA or FBS)

  • Centrifuge at 300g for 5 minutes and resuspend in 200 μL buffer for analysis

• Immunocytochemistry preparation:

  • Culture cells on appropriate substrates (coverslips, chamber slides)

  • Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

  • If detecting intracellular PRCP, permeabilize gently (0.1-0.2% Triton X-100, 5-10 minutes)

  • Block with 5-10% normal serum or 3-5% BSA (30-60 minutes)

  • Apply PRCP-FITC antibody at recommended dilution (1:10-50)

  • Incubate 1-2 hours at room temperature or overnight at 4°C in the dark

  • Wash thoroughly and mount with anti-fade medium containing nuclear counterstain

• Critical considerations:

  • Temperature control: Maintain 4°C during antibody incubation for flow cytometry to minimize internalization

  • Light protection: Shield FITC-conjugated antibodies from light to prevent photobleaching

  • Storage: Never freeze PRCP-FITC antibody as specified in the product information

  • Dilution optimization: Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Blocking buffer selection: Optimize to minimize background while preserving specific binding

These methodological details ensure consistent, specific staining and reliable experimental results across different applications.

What quantification methods should be used for PRCP-FITC signals in different applications?

Accurate quantification of PRCP expression using FITC-conjugated antibodies requires application-specific approaches:

• Flow cytometry quantification:

  • Mean/Median Fluorescence Intensity (MFI): Primary metric for quantifying PRCP expression level per cell

  • Percent positive cells: Determine the percentage of cells expressing PRCP above threshold

  • Standardized units: Convert to Molecules of Equivalent Soluble Fluorochrome (MESF) using calibration beads

  • Population analysis: Identify and separately analyze PRCP expression in distinct cell subsets

  • Statistical comparison: Use appropriate tests (t-test, ANOVA) to determine significant differences between samples

• Fluorescence microscopy quantification:

  • Integrated Density: Sum of pixel values in defined regions of interest

  • Corrected Total Cell Fluorescence (CTCF) = Integrated Density - (Area × Mean background fluorescence)

  • Cell-by-cell analysis: Measure PRCP expression in individual cells rather than field averages

  • Colocalization analysis: Quantify PRCP colocalization with other proteins using Pearson's or Mander's coefficients

  • Use software like ImageJ, CellProfiler, or MetaMorph for standardized analysis

• FLISA quantification:

  • Standard curve generation: Use known concentrations of recombinant PRCP

  • Sample interpolation: Determine unknown PRCP concentrations from standard curve

  • Use recommended 1:1000 dilution as starting point for optimization

  • Always run technical triplicates to account for pipetting variability

• Western blot quantification (for validation):

  • Use 1:100-500 dilution of PRCP-FITC antibody or unconjugated version

  • Normalize PRCP band intensity to loading control (β-actin, GAPDH)

  • Employ fluorescence scanning for more accurate quantification than chemiluminescence

These quantification approaches provide reliable methods to measure PRCP expression across experimental platforms, enabling meaningful comparisons between conditions or treatments.

How can researchers troubleshoot weak or non-specific PRCP-FITC antibody signals?

When encountering weak or non-specific PRCP-FITC antibody signals, systematic troubleshooting can identify and resolve the underlying issues:

• Weak signal troubleshooting:

  • Antibody concentration: Increase concentration within recommended ranges (FLISA: 1:1000; Western Blot: 1:100-500; Immunochemistry: 1:10-50)

  • Incubation conditions: Extend incubation time or optimize temperature

  • Epitope retrieval: For fixed samples, test different antigen retrieval methods

  • Signal amplification: Consider using anti-FITC secondary antibodies for signal enhancement

  • Sample handling: Ensure protein degradation is minimized during preparation

  • Instrument settings: Increase detector voltage/gain within linear range

• High background/non-specific signal troubleshooting:

  • Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers)

  • Washing stringency: Increase number or duration of washes

  • Autofluorescence: Include unstained controls and consider autofluorescence quenching methods

  • Antibody dilution: Test higher dilutions to reduce non-specific binding

  • Buffer optimization: Add 0.05-0.1% Tween-20 to reduce hydrophobic interactions

  • Fc receptor blocking: For immune cells, use Fc receptor blocking reagents

• Specificity verification:

  • Peptide competition: Pre-incubate antibody with immunizing peptide (N-terminal PRCP)

  • Knockdown validation: Compare signal between control and PRCP-depleted samples

  • Isotype control: Compare signal to rabbit IgG-FITC at identical concentration

  • Absorption controls: Pre-absorb antibody with cells known to express PRCP

• Technical considerations:

  • Storage conditions: Verify the antibody hasn't been frozen, as specified in product information

  • Light exposure: Minimize to prevent photobleaching of FITC conjugate

  • Sample viability: Exclude dead cells which can bind antibodies non-specifically

  • Spectral overlap: In multicolor experiments, ensure proper compensation

Systematic application of these troubleshooting approaches can resolve most technical issues with PRCP-FITC antibody staining.

How does PRCP antibody performance compare in different cancer cell models?

PRCP antibody performance and the biological insights it provides vary across cancer cell models, reflecting different roles of PRCP in various cancer types:

• Pancreatic cancer models:

  • Cell lines like Panc-1, PK-9, and Capan-1 show detectable PRCP expression

  • PRCP antibody staining correlates with IRS-1 stability and PI3K/AKT activation

  • In these models, PRCP/PREP depletion or inhibition reduces cell viability in a dose-dependent manner

  • PI3K inhibition with LY294002 or wortmannin produces similar effects to PRCP depletion

  • PRCP antibody can detect changes in expression following rapamycin treatment

• Breast cancer models (referenced in research background):

  • PRCP regulates proliferation and survival in breast cancer cells

  • Performance of PRCP antibody in breast cancer models would likely reveal similar regulatory mechanisms

• Experimental considerations across models:

  • Antibody dilution optimization: Different cell types may require adjusted dilutions from the standard recommendations (FLISA: 1:1000; Western Blot: 1:100-500; Immunochemistry: 1:10-50)

  • Background levels: Cancer cells often have higher autofluorescence requiring more stringent controls

  • Subcellular localization: PRCP distribution patterns may vary between cancer types

  • Correlation with pathway activity: PRCP antibody staining should be analyzed alongside PI3K/AKT pathway markers for functional correlation

• Methodological adaptations:

  • Sample preparation: Different cancer cell types may require optimized fixation/permeabilization protocols

  • Antigen preservation: Some cancer models may require specific buffers to preserve PRCP epitopes

  • Multiplexing strategies: Combining PRCP-FITC with markers relevant to specific cancer types

Understanding these model-specific considerations enables researchers to optimize PRCP antibody applications across diverse cancer research contexts.

What alternative molecular techniques complement PRCP antibody studies?

While PRCP antibody detection provides valuable information about protein expression and localization, complementary approaches offer deeper insights into PRCP biology:

• Genetic manipulation approaches:

  • RNA interference: siRNA/shRNA knockdown of PRCP for transient loss-of-function studies

  • CRISPR-Cas9 gene editing: Generate PRCP knockout cell lines for complete loss-of-function studies

  • Overexpression systems: Express tagged or untagged PRCP to study gain-of-function effects

  • Rescue experiments: Reintroduce wild-type or mutant PRCP into depleted cells to identify critical domains

• Enzymatic and functional assays:

  • PI3K activity assays: Measure PI3K activity in immunoprecipitates following PRCP manipulation

  • IRS-1 stability assays: Determine IRS-1 half-life using cycloheximide chase experiments

  • Phosphorylation analysis: Examine site-specific phosphorylation of IRS-1 at Ser-307 and Ser-636/639

  • Cell viability and apoptosis assays: MTT assays and apoptosis measurements to assess functional outcomes

• Pharmacological approaches:

  • PRCP/PREP inhibition with ZPP (Z-Pro-Prolinal)

  • Combination studies with pathway inhibitors like rapamycin (mTOR) or LY294002 (PI3K)

  • Dose-response analyses to establish inhibition parameters

  • Time-course studies to determine temporal relationships in signaling

• Molecular interaction studies:

  • Co-immunoprecipitation: Identify proteins physically interacting with PRCP

  • Proximity ligation assay: Visualize protein-protein interactions in situ

  • FRET/BRET analysis: Study dynamic interactions between PRCP and potential partners

These complementary approaches, when combined with PRCP antibody studies, provide a comprehensive understanding of PRCP's role in cellular signaling and its potential as a therapeutic target in cancer.