LAP3 Antibody

What is LAP3 and what cellular functions does it perform?

LAP3 (Leucine Aminopeptidase 3) is a cytosolic metallopeptidase belonging to the M17 aminopeptidase family that catalyzes the removal of unsubstituted N-terminal hydrophobic amino acids, particularly leucine, from various peptides . It plays key roles in:

• Protein degradation and peptide metabolism

• Glutathione metabolism and degradation of glutathione S-conjugates

• Cellular redox status regulation

• Cell proliferation, angiogenesis, and malignant development in various tissues

The enzymatic activity of LAP3 requires Zn²⁺ ions, while the association with other cofactors like Mn²⁺ can modulate its substrate specificity .

What applications are LAP3 antibodies commonly used for in research?

LAP3 antibodies are validated for multiple research applications:

Most researchers begin with Western blot to confirm antibody specificity before proceeding to more complex applications like IHC or immunofluorescence.

What are the recommended protocols for sample preparation in LAP3 immunohistochemistry?

For optimal LAP3 detection in tissue samples via IHC:

• Fix tissues in phosphate-buffered neutral formalin and embed in paraffin

• Section tissues to 5-μm thickness

• Perform heat-mediated antigen retrieval, preferably with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 can be used)

• Block with 10% BSA to reduce non-specific binding

• Incubate with primary LAP3 antibody (typically 1:50-1:400 dilution) overnight at 4°C

• Detect with appropriate secondary antibody conjugated to HRP

• Develop with diaminobenzidine and counterstain with hematoxylin

Proper antigen retrieval is critical, as many commercial antibodies show significantly improved staining after this step .

How do I validate LAP3 antibody specificity in my experiments?

A multi-step validation approach is recommended:

• Western blot validation: Compare observed band size (56 kDa) with predicted molecular weight. Use known positive control samples (e.g., HepG2, HeLa, or NIH/3T3 cells)

• Knockout controls: When available, use LAP3 knockout cell lines as negative controls. For example, Human LAP3 knockout A549 cell line has been successfully used to validate antibody specificity

• Peptide competition assay: Pre-incubate the antibody with blocking peptides containing the epitope recognized by the antibody to confirm binding specificity

• Cross-reactivity assessment: Test antibody against multiple species samples if cross-species reactivity is claimed or needed

• Multiple antibody comparison: When possible, compare results using antibodies targeting different epitopes of LAP3

What molecular mechanisms explain LAP3's role in cancer progression?

LAP3 contributes to cancer progression through multiple mechanisms:

• Cell cycle regulation: LAP3 promotes cancer cell proliferation by regulating the G1/S checkpoint. In hepatocellular carcinoma (HCC), LAP3 increases expression of cell cycle proteins including PCNA, cyclin A, CDK2, and CDK6

• HDAC2 upregulation: In breast cancer models, LAP3 upregulates histone deacetylase 2 (HDAC2) expression, which promotes cell cycle proteins cyclin A1 and D1 expressions, driving malignant transformation

• Arginine metabolism disruption: LAP3 downregulates argininosuccinate synthetase (ASS1), leading to arginine depletion, which contributes to metabolic reprogramming in cancer cells

• Enhanced migration: LAP3 advances migration capabilities of cancer cells, partly through modulating E-cadherin expression

• Chemoresistance: Knockdown of LAP3 enhances sensitivity of HCC cells to cisplatin, suggesting its role in drug resistance mechanisms

Clinical samples confirm LAP3 upregulation in multiple cancer types, including breast cancer and HCC, where its expression correlates with lower differentiation, positive lymph node metastasis, and high Ki-67 expression, indicating poor prognosis .

How does LAP3 interact with inflammatory pathways in disease conditions?

LAP3 has emerging roles in inflammatory processes:

• IFN-γ signaling: LAP3 is regulated by p38 and ERK MAPKs downstream of IFN-γ signaling. This regulation is crucial for LAP3's effects on arginine metabolism and cellular transformation

• SARS-CoV-2 infection response: LAP3 gene expression is significantly upregulated along with other inflammatory cytokines and chemokines following SARS-CoV-2 infection, suggesting its role in regulating viral inflammation responses

• NAFLD inflammation: In nonalcoholic fatty liver disease, cholesterol induces LAP3 upregulation, which subsequently inhibits autophagy, contributing to the inflammatory pathogenesis of NAFLD

To study these interactions effectively, researchers should:

• Examine LAP3 expression in response to various inflammatory stimuli

• Analyze phosphorylation status of MAPK pathways when manipulating LAP3 levels

• Use phospho-specific antibodies to track activation of inflammatory signaling cascades

• Consider cytokine profiling in LAP3 knockdown or overexpression models

What experimental approaches are most effective for studying LAP3's metabolic functions?

To investigate LAP3's metabolic roles:

• Targeted metabolomics:

  • Liquid chromatography-mass spectrometry (LC-MS) to measure specific metabolites affected by LAP3, particularly amino acids and glutathione-related compounds

  • Monitor arginine levels and related metabolites in LAP3 manipulated systems

• Enzymatic activity assays:

  • Measure LAP3 enzymatic activity using fluorogenic substrates

  • Assess effects of different metal ions (Zn²⁺, Mn²⁺) on substrate specificity

  • Determine kinetic parameters (Km, Vmax) under various conditions

• Gene manipulation strategies:

  • Use short hairpin RNA (shRNA) for LAP3 knockdown (example sequence: 5'-GCTCGGGCTCTATGAGTATGA-3')

  • Perform overexpression using vectors like pLenti-CMV-LAP3-GFP-Puro

  • Apply CRISPR-Cas9 for complete knockout models

• Redox status measurement:

  • Analyze GSSG/GSH ratio in cells with modified LAP3 expression

  • Measure intracellular reactive oxygen species (ROS) using fluorescent probes

  • Assess autophagy markers (e.g., LC3) to investigate LAP3's impact on cellular quality control

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify LAP3 binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry-based interactome analysis

How can researchers effectively study LAP3's role in drug resistance mechanisms?

LAP3 has been implicated in chemoresistance, particularly in hepatocellular carcinoma. To investigate this function:

• Cell viability assays:

  • Perform MTT or CCK-8 assays to compare drug sensitivity between LAP3 knockdown, overexpression, and control cells

  • Use flow cytometry to assess apoptosis rates following drug treatment

• Mechanistic studies:

  • Examine pathways associated with drug resistance (e.g., ABC transporters, anti-apoptotic proteins)

  • Monitor glutathione levels, as LAP3's role in glutathione metabolism may affect detoxification capacity

  • Investigate LAP3's impact on DNA damage repair mechanisms

• Gene expression profiling:

  • Conduct RNA-seq or qPCR arrays to identify expression changes in drug resistance genes following LAP3 manipulation

  • Analyze transcriptional changes in response to drug treatment in cells with altered LAP3 expression

• In vivo models:

  • Develop xenograft models with LAP3 knockdown or overexpression

  • Assess tumor response to chemotherapy in these models

  • Consider patient-derived xenografts to better model clinical relevance

• Clinical correlation studies:

  • Examine LAP3 expression in patient samples before and after treatment

  • Correlate expression levels with treatment outcomes and survival data

Example protocol for testing cisplatin sensitivity:

• Transfect cells with LAP3 siRNA or overexpression vector

• After 48 hours, treat with varying concentrations of cisplatin (0-100 μM)

• Measure cell viability at 24, 48, and 72 hours post-treatment

• Perform Western blot analysis for apoptosis markers (cleaved PARP, caspases)

What methods are recommended for investigating LAP3's tissue-specific functions in development and disease?

For tissue-specific LAP3 function analysis:

• Spatiotemporal expression profiling:

  • Perform immunohistochemistry on tissue arrays to compare LAP3 expression across multiple tissues

  • Use developmental series to track expression changes during organ maturation

  • Apply single-cell RNA sequencing to identify cell-specific expression patterns

• Tissue-specific knockout models:

  • Generate conditional knockout mice using Cre-loxP system targeting specific tissues

  • Analyze developmental impacts and disease susceptibility

  • Consider inducible systems to study temporal effects

• Organ-specific disease models:

  • For muscle development: Use C2C12 myoblast differentiation models and analyze effects of LAP3 manipulation on myogenic regulatory factors

  • For liver disease: Apply high-fat diet or cholesterol supplementation in LAP3 knockout mice to study NAFLD development

  • For cancer: Generate tissue-specific LAP3 overexpression models to assess oncogenic potential

• Ex vivo tissue culture:

  • Establish organoid cultures from tissues of interest

  • Manipulate LAP3 levels via viral transduction or CRISPR editing

  • Assess tissue-specific phenotypes and functions

• Functional readouts:

  • For muscle tissue: Analyze fusion index, myotube formation, and contractile properties

  • For liver: Measure lipid accumulation, inflammatory markers, and fibrosis

  • For epithelial tissues: Assess proliferation, polarity, and differentiation markers

What factors affect LAP3 antibody performance in different experimental contexts?

LAP3 antibody performance can be influenced by:

• Epitope accessibility:

  • Fixation methods significantly impact epitope preservation

  • Antigen retrieval is critical for many LAP3 antibodies in IHC applications

  • Different LAP3 antibodies may recognize distinct epitopes, affecting performance in native vs. denatured conditions

• Species cross-reactivity:

  • Many LAP3 antibodies show reactivity across human, mouse, and rat samples

  • Some antibodies have predicted reactivity for zebrafish, bovine, horse, sheep, and other species

  • Always validate antibodies for specific species applications

• Application-specific considerations:

  • For Western blot: Reducing conditions may affect epitope recognition

  • For IHC: Paraffin embedding vs. frozen sections require different antibody dilutions

  • For flow cytometry: Permeabilization methods impact intracellular staining quality

• Sample preparation:

  • Protein extraction methods affect yield and epitope integrity

  • Sample buffer composition influences antibody binding efficiency

  • Blocking agents must be optimized to reduce background without compromising specific binding

How can researchers troubleshoot non-specific binding or weak signals with LAP3 antibodies?

When encountering issues with LAP3 antibody performance:

• For weak signals:

  • Optimize antibody concentration (titration experiments)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance antigen retrieval (test different buffers and conditions)

  • Increase protein loading for Western blots

  • Try signal amplification systems (TSA, ABC method)

• For non-specific binding:

  • Increase blocking time and concentration (5-10% BSA or serum)

  • Add detergents to reduce hydrophobic interactions (0.1-0.3% Triton X-100)

  • Use more stringent washing conditions

  • Pre-adsorb antibody with non-specific proteins

  • Test alternative secondary antibodies

• For Western blot issues:

  • Verify transfer efficiency with reversible stains

  • Optimize blocking conditions (milk vs. BSA)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Reduce washing stringency if signal is weak

  • Consider enhanced chemiluminescence systems for detection

• For IHC optimization:

  • Compare different fixatives (formalin, paraformaldehyde)

  • Test multiple antigen retrieval methods (heat, enzymatic)

  • Use positive control tissues known to express LAP3

  • Consider amplification systems for low-abundance targets

  • Reduce endogenous peroxidase activity with hydrogen peroxide treatment

What are the most reliable positive control samples for LAP3 antibody validation?

Based on published research, reliable positive controls include:

• Cell lines:

  • HepG2 (human liver cancer cell line)

  • HeLa (human cervical cancer cell line)

  • 293T (human embryonic kidney cells)

  • NIH/3T3 (mouse fibroblast cells)

  • C2C12 (mouse myoblast cells)

• Tissue samples:

  • Human liver tissue (particularly hepatocellular carcinoma)

  • Human kidney tissue

  • Human ovarian carcinoma tissue

  • Human bladder transitional carcinoma

• Recombinant proteins:

  • Purified recombinant LAP3 protein (for Western blot optimization)

  • LAP3 fusion proteins with tags for antibody calibration

• Overexpression systems:

  • Cells transfected with pLenti-CMV-LAP3-GFP-Puro or similar vectors

  • Tissue lysates from LAP3 transgenic animals

These controls should be accompanied by appropriate negative controls, including LAP3 knockout cell lines (e.g., LAP3 knockout A549 cell line) or cells treated with LAP3-specific shRNA.

How should LAP3 antibodies be stored and handled to maintain optimal performance?

For maximum antibody stability and performance:

• Storage conditions:

  • Store at -20°C for long-term preservation

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

  • Some formulations contain glycerol (typically 50%) and can be stored at -20°C without aliquoting

  • Follow manufacturer's specific recommendations, which may vary by antibody

• Working solution handling:

  • Keep on ice when in use

  • Return to 4°C for short-term storage (typically stable for 1-2 weeks)

  • Avoid contamination by using clean pipette tips

  • Some antibodies may contain 0.02% sodium azide as preservative

• Dilution considerations:

  • Dilute in appropriate buffer (typically PBS with BSA)

  • Some formulations already contain 0.1% BSA for stabilization

  • Prepare fresh working dilutions when possible

  • For long-term experiments, consider adding preservatives to diluted antibody

• Quality control:

  • Monitor performance over time with consistent positive controls

  • Document lot numbers and observe for lot-to-lot variations

  • Consider antibody validation methods if performance declines

  • Check expiration dates and storage history if problems arise