APEH Antibody

What is APEH and why is it significant in research?

APEH (Acylpeptide hydrolase) is a protective protein produced by the immune system that catalyzes the hydrolysis of the N-terminal peptide bond of N-acetylated peptides, generating an N-acetylated amino acid and a peptide with a free N-terminus . APEH is significant in research because it plays important roles in:

• Destroying oxidatively damaged proteins in living cells

• Serving as a malaria parasite-internalized host enzyme that activates prodrugs

• Functioning in cellular metabolism and protein regulation

What are the main types of APEH antibodies available for research?

APEH antibodies come in several forms, each with specific research applications:

What is the difference between APEH and APE1 antibodies?

While both abbreviations appear similar, they target entirely different proteins:

• APEH antibodies target acylpeptide hydrolase, which functions in protein degradation and regulation

• APE1 antibodies target apurinic/apyrimidinic endonuclease 1, which plays a central role in DNA repair and redox regulation of transcriptional factors

These proteins have different molecular weights, cellular localizations, and biological functions, requiring specific validation for their respective antibodies.

How should I validate an APEH antibody for my specific research application?

Comprehensive validation should follow these methodological steps:

• Preliminary verification: Check species reactivity in product documentation (human APEH antibodies may cross-react with mouse (88%), rat (89%), and other species)

• Positive control selection: Use recommended positive controls like HeLa cell lysates

• Application-specific validation:

  • For Western blot: Verify band at approximately 80 kDa (erythrocyte form) or 55 kDa (parasite-internalized form)

  • For immunofluorescence: Compare with published localization patterns (cytoplasmic in erythrocytes, but can be internalized by P. falciparum)

• Knockdown/knockout validation: If possible, use siRNA or CRISPR techniques to reduce APEH expression as negative controls

What are the optimal working dilutions for different APEH antibody applications?

Based on manufacturer recommendations across multiple sources:

How can I optimize immunofluorescence protocols specifically for APEH detection in malaria-infected erythrocytes?

Based on published protocols for APEH detection in P. falciparum:

• Fixation: Use a combination of paraformaldehyde and glutaraldehyde to preserve both parasite and erythrocyte structures

• Blocking: Block with PBST + 3% BSA for 1 hour at room temperature

• Primary antibody: Use rabbit anti-APEH antibodies at dilutions from 1:100 to 1:900 in PBST + 3% BSA

• Incubation: Incubate for 1 hour at room temperature

• Washing: Wash thoroughly with PBST to remove unbound antibody

• Secondary detection: Use fluorescent-labeled secondary antibodies (e.g., Goat anti-Rabbit IgG Alexa Fluor 488)

• Nuclear staining: Counterstain with 2μM Hoechst for 10 minutes

• Imaging: Image using appropriate fluorescence microscopy (e.g., Nikon Eclipse Ti2 microscope)

• Mounting: Plate cells on Poly-D-Lysine coated μ-Plate for optimal adhesion

How can I distinguish between erythrocyte APEH and parasite-internalized APEH in malaria research?

The distinction can be made through several complementary approaches:

• Molecular weight analysis: Erythrocyte APEH appears as an 80-kDa species while parasite-internalized APEH shows a 55-kDa form in Western blots

• Selective inhibition: Use AA74-1 at appropriate concentrations to selectively inhibit APEH activity:

  • IC₅₀ values: 7.9 ± 1.8 nM for parasite 55-kDa species and 7.4 ± 2.4 nM for erythrocyte 80-kDa species

• Localization studies: Use immunofluorescence with APEH antibodies to visualize:

  • Normal distribution in uninfected erythrocytes

  • Internalization into parasite cytoplasm in infected erythrocytes

• Functional assays: Measure activation of APEH-specific substrates in parasite lysates with and without AA74-1 treatment

What experimental controls are essential when using APEH antibodies to study prodrug activation mechanisms?

When investigating APEH's role in prodrug activation, include these critical controls:

• Enzyme inhibition controls:

  • Pre-treat samples with APEH inhibitor AA74-1 (100 nM) to confirm APEH-specific activity

  • Include fosmidomycin as a control that doesn't require APEH activation

• Substrate specificity controls:

  • Test multiple substrates with different structural features

  • Include substrates with branching at alpha carbon (APEH preference) and long-chain unbranched alkyl esters (minimal APEH activation)

• Enzyme source controls:

  • Compare purified recombinant APEH (rAPEH)

  • Use P. falciparum lysate and erythrocyte lysate

  • Test parasite lysate pre-treated with AA74-1

• Activity measurement controls:

  • Direct measurement of prodrug activation products

  • Indirect measurement through downstream enzyme activities (e.g., DXR activity assay)

How should I interpret discrepancies in APEH antibody labeling patterns between different detection methods?

When faced with conflicting results across methods:

• Antibody epitope consideration: Different antibodies may recognize distinct epitopes - some manufacturers cite antibodies targeting amino acids 1-260 of human APEH, while others may target different regions

• Protein processing analysis: APEH exists in different forms:

  • Full-length (~80 kDa) in erythrocytes

  • Processed form (~55 kDa) in parasites

  • Potential post-translational modifications

• Methodological differences:

  • Western blot detects denatured proteins

  • IF/IHC detect proteins in their native conformation and cellular context

  • Cross-reactivity may differ between applications

• Resolution approach: Use multiple antibodies from different manufacturers and clones, targeting different epitopes, to validate your findings

What storage conditions are critical for maintaining APEH antibody quality and performance?

Optimal storage practices include:

• Temperature: Store concentrated antibody solutions at -20°C to maintain activity

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Formulation: Most APEH antibodies are provided in buffer containing:

  • PBS (pH 7.3)

  • 50% glycerol as cryoprotectant

  • Some may contain preservatives like 0.03% Proclin 300

• Preparation before use: Centrifuge briefly prior to opening the vial

• Working dilution storage: Store diluted working solutions at 4°C for short-term use only (1-2 weeks)

How can I address non-specific binding issues when using APEH antibodies in complex samples?

When encountering background or non-specific signals:

• Blocking optimization:

  • Test different blocking agents (BSA, milk, normal serum from secondary antibody host)

  • Increase blocking time (from 1 hour to overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 for improved penetration in IF/IHC

• Antibody dilution adjustment:

  • Increase dilution if background is high

  • Consider titrating antibody to determine optimal signal-to-noise ratio

• Washing procedures:

  • Increase number of washes (5-6 times)

  • Extend washing duration (10-15 minutes per wash)

  • Add low concentrations of Tween-20 (0.05-0.1%) to wash buffers

• Pre-adsorption:

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

  • Consider pre-adsorbing antibody with tissue powder from relevant species

How should I validate APEH antibody specificity when working with multi-species samples or conserved proteins?

For cross-species applications:

• Sequence homology analysis:

  • Compare APEH sequences across target species (e.g., human APEH shows 88% homology with mouse, 89% with rat)

  • Identify conserved epitopes that antibodies may recognize

• Western blot validation:

  • Run lysates from multiple species side-by-side

  • Verify appropriate molecular weight shifts based on species differences

  • Use positive controls with known APEH expression

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be abolished if antibody is specific

  • Include gradient of peptide concentrations

• Genetic validation:

  • When possible, use samples from APEH knockout models

  • Alternatively, use siRNA knockdown in cell lines from different species

How can APEH antibodies be used to investigate malaria drug resistance mechanisms?

APEH antibodies enable several approaches to studying antimalarial drug resistance:

• Prodrug activation analysis:

  • Compare APEH uptake in drug-sensitive vs. resistant parasite strains

  • Quantify APEH levels in parasites using calibrated Western blot or quantitative immunofluorescence

  • Correlate APEH activity with prodrug efficacy across clinical isolates

• Localization studies:

  • Use immunofluorescence to track changes in APEH localization in resistant strains

  • Determine if resistant parasites alter APEH trafficking within the parasite

• Structure-activity relationship studies:

  • Use purified APEH (identified via antibodies) to test activation of different prodrug candidates

  • Develop prodrugs specifically activated by internalized host APEH to circumvent parasite resistance mechanisms

• Intervention strategies:

  • Target pathways that modulate APEH internalization

  • Use selective inhibitors that affect parasite utilization of host APEH without inhibiting host APEH function

What methodological approaches can I use to study APEH's role in oxidative stress responses?

APEH's function in processing oxidatively damaged proteins can be investigated through:

• Co-localization studies:

  • Use APEH antibodies alongside markers of oxidative stress

  • Quantify spatial correlation between APEH and oxidized proteins

• Functional analysis:

  • Measure APEH activity (using fluorogenic substrates) before and after oxidative stress induction

  • Correlate with protein oxidation markers using OxyBlot or similar techniques

• Proteomic approaches:

  • Immunoprecipitate APEH using validated antibodies

  • Identify binding partners and substrates by mass spectrometry

  • Compare APEH-associated proteins under normal vs. oxidative stress conditions

• Genetic manipulation:

  • Overexpress or knock down APEH in cell models

  • Measure cell viability and oxidative damage markers after stress challenge

  • Use APEH antibodies to confirm expression changes

What experimental design considerations are important when investigating APEH in neurodegenerative disease models?

When exploring APEH's potential role in neurodegeneration:

• Tissue-specific validation:

  • Validate APEH antibodies specifically in neural tissues

  • Compare antibody performance in different brain regions

  • Optimize protocols for fixed brain tissue sections

• Age-dependent expression analysis:

  • Compare APEH levels across different age groups

  • Correlate with markers of protein aggregation or oxidative damage

  • Use both Western blot and immunohistochemistry for quantitative and spatial information

• Cell-type specific detection:

  • Perform double labeling with cell-type markers (neurons, astrocytes, microglia)

  • Use confocal microscopy to determine precise cellular localization

  • Consider single-cell analyses to detect population heterogeneity

• Disease model integration:

  • Compare APEH levels and localization in disease models vs. controls

  • Correlate with disease progression markers

  • Test APEH modulators as potential therapeutic approaches