XPNPEP1 Recombinant Monoclonal Antibody

What is XPNPEP1 and what biological function does it serve?

XPNPEP1 (X-prolyl aminopeptidase 1) is a cytosolic metalloaminopeptidase that catalyzes the cleavage of N-terminal amino acids adjacent to proline residues. This enzyme plays a significant role in the degradation and maturation of bioactive peptides including tachykinins, neuropeptides, and peptide hormones . The gene encoding XPNPEP1 is subject to alternative splicing, resulting in multiple transcript variants . Recent research has identified XPNPEP1 as a potential protective gene biomarker for Acute Coronary Syndrome (ACS), suggesting its relevance in cardiovascular pathology .

What applications have been validated for XPNPEP1 recombinant monoclonal antibodies?

XPNPEP1 recombinant monoclonal antibodies have been validated for multiple research applications with consistent performance across different experimental platforms:

Researchers should note that these antibodies are strictly for research use only and not approved for diagnostic procedures .

What are the optimal handling and storage conditions for XPNPEP1 antibodies?

To maintain optimal antibody performance and stability, XPNPEP1 recombinant monoclonal antibodies should be stored according to the following guidelines:

• Short-term storage (up to 3 months): Store at 4°C

• Long-term storage: Store at -20°C, avoiding freeze/thaw cycles

• Buffer composition: Typically supplied in PBS with 0.02% ProClin 300 as preservative

• Concentration: Usually provided at 1 mg/mL

• Aliquoting: Divide the antibody into small working aliquots before freezing to avoid repeated freeze/thaw cycles that can compromise antibody integrity

The stability of the antibody under these conditions allows for consistent experimental results over time when proper storage protocols are followed.

How can researchers verify the specificity of XPNPEP1 recombinant monoclonal antibodies?

Confirming antibody specificity is critical for experimental validity. For XPNPEP1 recombinant monoclonal antibodies, specificity can be verified through:

• Western blot analysis: Using cell lysates with known XPNPEP1 expression (e.g., HeLa, Jurkat, MCF7, HepG2, A549, HEK293) to confirm the detection of a single band at the expected molecular weight

• Immunofluorescence with blocking peptides: Performing parallel staining with and without pre-incubation with the immunizing peptide ("GSLTFEPLTLVPIQTK" for clone SAIC-07B-14)

• Flow cytometry on positive and negative control cell lines: Comparing staining profiles between cell lines with differential XPNPEP1 expression

• Knockout/knockdown validation: Using CRISPR/Cas9 knockout or siRNA knockdown cells to confirm loss of signal with XPNPEP1 depletion

These validation approaches should be documented in laboratory records to support the reliability of research findings.

What methodological considerations are important when using XPNPEP1 antibodies in cardiovascular research?

The identification of XPNPEP1 as a potential protective biomarker in Acute Coronary Syndrome highlights its significance in cardiovascular research. When designing experiments in this context, researchers should consider:

• Temporal expression dynamics: XPNPEP1 expression may vary following cardiac events; sample collection timing should be standardized (e.g., within 12 hours of symptom onset for ACS patients)

• Cell type-specific expression: Immunofluorescence staining protocols for cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have been established using anti-rabbit XPNPEP1 antibody (1:50 dilution)

• Comparative analysis framework: Include samples from both healthy donors and ACS patients to assess differential expression patterns

• Complementary validation: Combine antibody-based detection with quantitative RT-PCR to verify expression changes at both protein and mRNA levels

• Co-localization studies: When performing immunofluorescence, use cardiac-specific markers (e.g., cardiac troponin) alongside XPNPEP1 to confirm expression in cardiomyocytes

The standard protocol for hiPSC-CM staining includes fixation with 4% paraformaldehyde (1 hour), permeabilization with 1% Triton X-100 (1 hour), and staining with anti-XPNPEP1 antibody followed by fluorescent secondary antibody (goat anti-rabbit IgG Alexa Fluor 594, 1:100) .

How should researchers optimize antibody dilutions for different experimental applications?

Optimal antibody dilution varies by application and must be empirically determined to maximize signal-to-noise ratio. Based on available data for XPNPEP1 recombinant monoclonal antibodies:

Optimization strategy:

• Begin with manufacturer's recommended dilution

• Perform a dilution series experiment covering at least 3 concentrations

• Include positive controls (cells/tissues known to express XPNPEP1) and negative controls (secondary antibody only)

• Select the dilution that provides maximum specific signal with minimal background

For immunofluorescence applications specifically in cardiomyocytes, the validated dilution is 1:50 for the primary antibody and 1:100 for the secondary antibody .

What are the critical considerations when interpreting XPNPEP1 expression data in disease models?

When analyzing XPNPEP1 expression in pathological contexts, especially in ACS and other cardiovascular diseases, researchers should consider several factors:

• Biomarker context: XPNPEP1 has been identified as a potential protective gene biomarker for ACS , suggesting its expression levels may correlate with disease severity or prognosis

• Alternative transcript consideration: Due to alternative splicing resulting in multiple transcript variants , antibody epitope location should be verified to ensure detection of all relevant isoforms

• Integration with circRNA-related ceRNA networks: Recent studies have constructed competitive endogenous RNA networks in ACS involving XPNPEP1 , indicating the need to consider broader regulatory mechanisms

• Cell type heterogeneity: When analyzing tissue samples, consider that differential expression across cell types may obscure biologically significant changes in specific cell populations

• Temporal dynamics: Expression patterns may change during disease progression or in response to treatment, necessitating time-course experimental designs

Researchers should use multiple detection methods (antibody-based and nucleic acid-based) to comprehensively assess XPNPEP1 status in experimental and clinical samples.

How can XPNPEP1 antibodies be integrated into multiplexed detection systems?

For complex experimental designs requiring simultaneous detection of XPNPEP1 and other markers:

• Multiplex immunofluorescence considerations:

  • The SAIC-07B-14 clone has been validated for immunofluorescence , making it suitable for multiplex applications

  • When designing panels, consider antibody species origin (rabbit for SAIC-07B-14 ) to avoid cross-reactivity

  • For co-localization with cardiac markers, use species-distinct primary antibodies (e.g., mouse anti-cardiac troponin with rabbit anti-XPNPEP1)

• Mass spectrometry-based approaches:

  • The SAIC-07B-14 clone has been validated for immuno-MRM applications

  • For quantitative proteomics, consider immunoprecipitation with XPNPEP1 antibody followed by mass spectrometry analysis

  • Optimization of peptide extraction and digestion protocols is essential for detecting endogenous XPNPEP1

• Flow cytometry multi-parameter analysis:

  • XPNPEP1 antibodies have been validated for flow cytometry applications

  • For intracellular staining, optimize fixation and permeabilization conditions to maintain epitope accessibility

  • When analyzing cardiovascular samples, include markers for different cell populations (e.g., endothelial cells, cardiomyocytes, inflammatory cells)

What troubleshooting strategies should be employed when XPNPEP1 antibody experiments yield inconsistent results?

When facing experimental variability with XPNPEP1 antibodies, consider the following systematic approach:

• Antibody validation issues:

  • Verify antibody lot consistency through lot-specific validation experiments

  • Confirm epitope integrity by comparing results with antibodies targeting different XPNPEP1 regions

  • For clone SAIC-07B-14, verify recognition of the specific epitope "GSLTFEPLTLVPIQTK"

• Sample preparation variables:

  • Standardize cell lysis conditions to maintain protein conformation

  • For cardiovascular samples, minimize time between collection and processing to prevent degradation

  • Document freeze-thaw cycles of both samples and antibodies

• Technical optimization:

  • For western blotting: Adjust protein transfer conditions, blocking reagents, and detection systems

  • For immunofluorescence: Optimize fixation time, permeabilization conditions, and antigen retrieval methods

  • For flow cytometry: Fine-tune permeabilization protocol and antibody incubation temperature

• Biological variability assessment:

  • Consider donor-to-donor variability in primary cells

  • For hiPSC-derived cardiomyocytes, account for differentiation efficiency and maturation state

  • In ACS studies, stratify samples based on clinical parameters (STEMI, NSTEMI, UA) and time since symptom onset

A systematic investigation of these factors should identify sources of variability and allow for protocol optimization to achieve consistent results.

What emerging research areas might benefit from XPNPEP1 antibody applications?

The identification of XPNPEP1 as a potential protective biomarker in Acute Coronary Syndrome opens several promising research directions:

• Cardiovascular precision medicine: Investigating XPNPEP1 expression patterns across ACS patient subgroups may help stratify patients for tailored therapeutic approaches

• Drug development: Screening compounds that modulate XPNPEP1 expression or activity might yield novel cardioprotective agents

• Biomarker development: Further validation of XPNPEP1 in larger patient cohorts could establish its utility as a diagnostic or prognostic marker in cardiovascular disease

• Mechanistic studies: Investigating the precise role of XPNPEP1 in cardiovascular protection may reveal new pathways for therapeutic intervention

• Regenerative medicine: Exploring XPNPEP1 function in hiPSC-derived cardiomyocytes may contribute to understanding cardiac repair and regeneration mechanisms