NPEPL1 Antibody

What is NPEPL1 and what biological functions does it serve?

NPEPL1 (Aminopeptidase-Like 1) is a 523 amino acid protein belonging to the peptidase M17 family. It plays a crucial role in processing and degrading intracellular proteins by catalyzing the removal of unsubstituted N-terminal amino acids from various peptides. This function maintains cellular homeostasis and regulates protein turnover, which is essential for various physiological processes, including hormone regulation and metabolic pathways . The protein features several zinc-binding sites important for enzymatic activity and exists in three isoforms due to alternative splicing events, allowing for functional diversity in different cellular contexts .

What are the optimal applications for NPEPL1 antibody detection?

NPEPL1 antibodies have been validated for multiple applications with varying degrees of efficacy:

For optimal results, researchers should select antibodies specifically validated for their intended application. For instance, Proteintech's 17211-1-AP antibody is validated for WB, IHC, IF/ICC, and ELISA with human, mouse, and rat samples .

What is the recommended working dilution for NPEPL1 antibodies?

Optimal dilutions vary by antibody and application:

It is strongly recommended to titrate each antibody in your specific experimental system to determine optimal conditions, as sample type and preparation methods can significantly impact antibody performance .

What cellular and tissue distribution patterns are observed for NPEPL1?

NPEPL1 is ubiquitously expressed across multiple tissue types. Immunohistochemistry studies have successfully detected NPEPL1 in human kidney tissue, suggesting expression in renal tissues . At the cellular level, NPEPL1 has been detected in various cell lines, including HeLa and MCF-7 cells using Western blotting and immunofluorescence techniques . The expression pattern can vary between normal and pathological states, with upregulation observed in certain cancer tissues, particularly clear cell renal cell carcinoma (ccRCC) .

How does NPEPL1 expression correlate with cancer progression and prognosis?

Studies using The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases have revealed significant correlations between NPEPL1 expression and cancer outcomes:

Beyond ccRCC, NPEPL1 has been implicated in prostate, breast, and colorectal cancers, suggesting a broader role in oncogenic processes .

What signaling pathways and molecular mechanisms are influenced by NPEPL1?

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses have identified several mechanisms potentially mediated by NPEPL1:

• Cellular components: Enrichment in voltage-gated calcium channel complex and immunoglobulin complex .

• Molecular functions: Channel activity and passive transmembrane transporter activity .

• Signaling pathways: Involvement in cAMP signaling pathway, oxytocin signaling pathway, neuroactive ligand-receptor interaction, and pancreatic secretion pathways .

• Biological processes: Detection of external stimulus and detection of abiotic stimulus .

Differential gene expression analysis comparing high and low NPEPL1 expression identified 5,679 differentially expressed genes (5,635 upregulated, 44 downregulated), suggesting NPEPL1 may function within complex gene regulatory networks .

How does NPEPL1 influence tumor immune microenvironment and drug sensitivity?

NPEPL1 expression appears to modulate both immune cell infiltration and therapeutic responses:

Immune microenvironment effects:

• "CIBERSORT" analysis revealed that elevated NPEPL1 expression correlates with enrichment of regulatory T cells and follicular helper T cells in the tumor microenvironment .

• This suggests NPEPL1 may influence immunosuppressive mechanisms within tumors, potentially affecting immunotherapy responses.

Drug sensitivity correlations:

• Patients with high NPEPL1 expression demonstrated increased sensitivity to specific therapeutics:

  • Greater benefit from axitinib (tyrosine kinase inhibitor)

  • Enhanced response to cisplatin (platinum-based chemotherapy)

  • Increased sensitivity to GSK429286A

These findings suggest NPEPL1 expression might serve as a biomarker for treatment selection, potentially guiding personalized therapeutic approaches in oncology.

What technical challenges exist in validating NPEPL1 antibody specificity?

Ensuring antibody specificity presents several methodological challenges:

• Cross-reactivity concerns: NPEPL1 belongs to the aminopeptidase family with structural similarities to other family members, increasing the risk of cross-reactivity. Researchers should validate specificity through knockout/knockdown controls.

• Isoform detection: NPEPL1 exists in multiple isoforms due to alternative splicing. Confirming which isoforms are detected by a specific antibody requires careful epitope mapping and validation .

• Species conservation: NPEPL1 shows high sequence conservation across species (up to 100% identity in many regions by BLAST analysis) . While this facilitates cross-species application, it may complicate distinguishing between orthologues in mixed-species experiments.

• Polyreactivity considerations: Recent research on antibody polyreactivity highlights that some antibodies demonstrate broader binding profiles than expected. Evaluating NPEPL1 antibodies for polyreactivity should be considered, especially in complex experimental systems .

What are the optimal sample preparation techniques for NPEPL1 antibody applications?

For Western Blotting:

• Optimal lysis buffers: RIPA buffer with protease inhibitors is generally effective

• Recommended protein loading: 20-40 μg total protein per lane

• Reduction conditions: Standard DTT or β-mercaptoethanol treatment

• Expected molecular weight: ~52 kDa observed (calculated MW: 56 kDa)

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin (24-48 hours)

• Antigen retrieval: TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Blocking: 3% BSA or 5-10% normal serum matching secondary antibody host

• Incubation: Overnight at 4°C for primary antibody

For Immunofluorescence:

• Fixation: 4% paraformaldehyde (15-20 minutes at room temperature)

• Permeabilization: 0.1-0.5% Triton X-100 (10 minutes)

• Blocking: 5% normal serum with 0.3% Triton X-100

• Mounting: Anti-fade mounting medium with DAPI for nuclear counterstain

How can researchers validate the specificity of NPEPL1 antibodies?

A comprehensive validation approach should include multiple strategies:

• Genetic validation:

  • siRNA/shRNA knockdown of NPEPL1 followed by Western blot

  • CRISPR/Cas9 knockout cell lines as negative controls

  • Overexpression systems as positive controls

• Biochemical validation:

  • Pre-adsorption with immunizing peptide/protein

  • Multiple antibodies targeting different epitopes

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Cross-platform validation:

  • Correlation of protein detection with mRNA expression

  • Comparison across multiple detection methods (WB, IHC, IF)

  • Testing in multiple cell lines with known NPEPL1 expression levels

• Controls to include:

  • Positive control: HeLa or MCF-7 cells (known to express NPEPL1)

  • Negative control: Primary antibody omission

  • Isotype control: Non-specific IgG matching the host species

What considerations should be made when selecting a NPEPL1 antibody for specific research applications?

When selecting a NPEPL1 antibody, researchers should evaluate:

Epitope characteristics:

• Location: Different antibodies target distinct regions (e.g., AA 405-454, AA 371-470, N-terminal, C-terminal)

• Conservation: Epitopes with 100% identity across species enable cross-species application

• Domain specificity: Consider whether specific functional domains need to be targeted

Technical specifications:

• Clonality: Polyclonal antibodies offer broader epitope recognition; monoclonal antibodies provide higher specificity

• Host species: Choose based on compatibility with experimental system and secondary antibodies

• Purification method: Immunoaffinity purified antibodies generally offer higher specificity

Validation extent:

• Application-specific validation: Ensure the antibody is validated for your specific application

• Publication record: Previously published studies using the antibody provide confidence in performance

• Lot-to-lot consistency: Evaluate manufacturer quality control data

Experimental compatibility:

• Buffer formulation: Consider compatibility with your experimental system

• Storage conditions: Evaluate stability requirements and aliquoting recommendations

• Sample type compatibility: Ensure validation in your specific sample type (human, mouse, etc.)

How can NPEPL1 antibodies be effectively used in multiplexed detection systems?

Implementing NPEPL1 antibodies in multiplexed systems requires careful optimization:

For multiplexed immunofluorescence:

• Antibody panel design:

  • Select NPEPL1 antibodies from different host species than other target antibodies

  • Consider using directly conjugated primary antibodies when possible

  • Test for cross-reactivity between antibodies in the panel

• Sequential staining approach:

  • When using same-species antibodies, consider tyramide signal amplification

  • Employ complete antibody stripping between rounds

  • Validate signal specificity after each round

• Spectral considerations:

  • Select fluorophores with minimal spectral overlap

  • Include appropriate controls for spectral unmixing

  • Validate staining patterns with single-color controls

For mass cytometry applications:

• Conjugate NPEPL1 antibodies with rare earth metals

• Validate signal preservation post-conjugation

• Include isotype controls labeled with the same metal

For multiplexed immunohistochemistry:

• Optimize chromogen selection for visual discrimination

• Consider automated staining platforms for consistency

• Validate each antibody independently before multiplexing

What is the recommended approach for quantifying NPEPL1 expression in tissue samples?

Quantitative assessment of NPEPL1 expression should follow these methodological principles:

For immunohistochemistry quantification:

• Scoring systems:

  • H-score: Combines intensity (0-3) and percentage of positive cells

  • Allred score: Sum of proportion score (0-5) and intensity score (0-3)

  • Automated image analysis: Software-based quantification of staining intensity and distribution

• Standardization approaches:

  • Include reference control slides in each batch

  • Use digital scanning for consistent analysis

  • Employ multiple independent scorers for validation

• Cutoff determination:

  • Establish thresholds for "high" vs. "low" expression based on:

    • Statistical methods (e.g., receiver operating characteristic curves)

    • Correlation with clinical outcomes

    • Comparison with normal tissue expression

For Western blot quantification:

• Use proper loading controls (e.g., GAPDH, β-actin)

• Employ densitometry with linear dynamic range validation

• Present data as relative expression normalized to controls

This analytical approach has been successfully applied to identify the prognostic value of NPEPL1 expression in ccRCC, where increased expression correlates with poorer outcomes across multiple clinical parameters .