enpp4 Antibody

Basic Research Questions

• What is ENPP4 and what key biological functions does it serve in research models?

  ENPP4 (ectonucleotide pyrophosphatase/phosphodiesterase 4) belongs to the enpp ectonucleotidase family that regulates lipidic and purinergic signaling pathways by controlling extracellular concentrations of purines and bioactive lipids . Functionally, ENPP4:

  • Hydrolyzes phosphodiester bonds in nucleotides with preference for adenine nucleotides

  • Cleaves diadenosine compounds Ap3A and Ap4a released from thrombin-activated platelets

  • Generates AMP/ADP from Ap3A cleavage and AMP/ATP from Ap4A cleavage

  • Prolongs platelet aggregation when expressed on vascular endothelial cells

  • Demonstrates tumoricidal effects against MCA207 cells when expressed on BCG-activated macrophages

  • Plays a role in kidney development, with overexpression resulting in ectopic pronephric tubules formation

• What is the tissue expression profile of ENPP4 and how should I detect it?

  ENPP4 expression varies significantly across tissues. In mice, ENPP4 is:

  • Abundantly expressed in spleen, stomach, and ovary

  • Not detectable in brain, lung, kidney, thymus, liver, heart, uterus, and intestine

  For detection, immunohistochemistry using specific anti-ENPP4 antibodies is recommended. Protocol guidance:

  1. Fix tissues in formalin at 4°C overnight

  2. Embed in paraffin and section for histology

  3. Inactivate endogenous peroxidase with 0.3% H₂O₂ (10 min, room temperature)

  4. Incubate with anti-ENPP4 primary antibody (1 hr, room temperature)

  5. Wash and incubate with peroxidase-conjugated secondary antibody (40 min)

  6. Detect signals using DAB (3,3-diaminobenzidine tetrahydrochloride)

• What are the key structural characteristics of ENPP4 relevant to antibody selection?

  ENPP4 is a 453 amino acid single-pass type I membrane protein with specific structural domains:

  • Full sequence of human ENPP4: MKLLVILLFSGLITGFRSDSSSLPPKLLLVSFDGFRADYLKNYEFPHLQNFIKEGVLVEHVKNVFITKTTPNHYSIVTGLYEESHGIVANSMYDAVTKKHFSDSNDKDPFWWNEAVPIWVTNQLQENRSSAAAMWPGTDVPIHDTISSYFMYNSSVSFERLNNITMWLNNSNPPVTFATLYWEEPDASGHKYGPEDKENMSRVLKKIDDLIGDLVQRLKMLGLWENLNVIITSDHGMTQCSQDRLINLDSCIDHSYYTLIDLSPVAAILPKINRTEVYNKLKNCSPHNVYLKDIPNRFYYQHNDRIQPIILVADEGWTIVLNESSQKLGDHGYDNSLPSMHPFLAAHGPAFHKGYKHSTINIVDIYPMMCHILGLKPHPNNGTFGHTKCLLVDQWCINLPEAIAIVIGLLVLTMLTCLIIMQNRLVPRPFSRLQLQEDDDDPLIG

  • Contains a catalytic domain with key residues (Arg305, Tyr341, Asn291, and Asn295) important for substrate (ATP) binding

  • Amino acids 171-200 represent a central region often targeted by antibodies

  • Includes zinc-binding regions necessary for enzymatic function

  When selecting antibodies, consider whether you need recognition of specific domains or the full-length protein based on your experimental questions.

• What experimental applications are ENPP4 antibodies validated for?

  ENPP4 antibodies have been validated for multiple applications:

  Application	Description	Typical Dilution
  Western Blotting (WB)	Detection of denatured ENPP4 protein (~51.6 kDa)	1:1000
  ELISA	Quantitative measurement of ENPP4 protein	Variable by kit
  Immunohistochemistry (IHC)	Tissue localization of ENPP4	1:100-1:500

  Selection should be based on the specific experiment type, species reactivity (human, mouse, rat), and the antibody's validated applications .

Advanced Research Questions

• How can I optimize immunohistochemistry protocols for detecting low-abundance ENPP4 in different tissue types?

  Optimizing IHC for ENPP4 requires special consideration for tissue-specific backgrounds and expression levels:

  1. Antigen retrieval optimization: For formalin-fixed tissues where ENPP4 detection is challenging, compare:

     • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

     • Trypsin-based enzymatic retrieval (0.05% for 20 minutes at 37°C)

     • EDTA buffer (pH 9.0) for exposing epitopes in highly fixed samples

  2. Signal amplification strategies:

     • Implement tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold

     • Use polymer-based detection systems rather than traditional ABC methods

     • Consider overnight primary antibody incubation at 4°C rather than 1 hour at room temperature

  3. Tissue-specific considerations:

     • For spleen, stomach, and ovary (high ENPP4 expressors): Use higher antibody dilutions (1:500-1:1000)

     • For potential low-expressors: Reduce dilution (1:50-1:100) and extend incubation times

     • Include appropriate positive controls (spleen sections) and negative controls

• What methodological approaches are most effective for studying ENPP4's tumoricidal activity in BCG-activated macrophages?

  To investigate ENPP4's role in tumoricidal activity:

  1. BCG activation of macrophages:

     • Immunize mice intraperitoneally with BCG (e.g., 4 mg) in multiple doses

     • Harvest peritoneal macrophages after the final immunization

     • Verify ENPP4 upregulation via Western blot or flow cytometry

  2. Contact-dependent cytotoxicity assay:

     • Fix BCG-activated macrophages with 1% paraformaldehyde (30 min, room temperature)

     • Block ENPP4 on separate samples using anti-ENPP4 antibodies

     • Co-culture with target tumor cells (e.g., MCA207) in 96-well plates

     • Assess tumor cell viability after 48 hours using MTT assay

  3. Direct protein activity assessment:

     • Purify recombinant ENPP4 protein

     • Add at various concentrations (1-40 μg/mL) to tumor cell cultures

     • Measure cytotoxic effects after 48 hours

     • Compare with antibody-blocking experiments to confirm specificity

  This approach allows comparison between blocked and unblocked ENPP4, providing direct evidence of ENPP4's contribution to the tumoricidal effect.

• How do I troubleshoot inconsistent ENPP4 antibody staining patterns across different experimental conditions?

  Inconsistent ENPP4 staining often stems from multiple factors:

  1. Antibody validation:

     • Confirm antibody specificity using positive controls (tissues with known ENPP4 expression like spleen)

     • Run Western blots to verify correct molecular weight detection (~51.6 kDa)

     • Test multiple antibody lots if possible

  2. Sample preparation issues:

     • Standardize fixation times (overfixation can mask epitopes)

     • Optimize antigen retrieval for each tissue type

     • Ensure consistent section thickness (4-5 μm recommended)

  3. Technical considerations:

     • Implement automated staining platforms to reduce variability

     • Use humidity chambers during incubations to prevent edge effects

     • Prepare fresh reagents, especially detection substrates like DAB

  4. Data analysis approach:

     • Blind scoring by multiple observers

     • Consider digital image analysis for objective quantification

     • Compare staining patterns with mRNA expression data if available

• What are the best approaches to validate ENPP4 antibody specificity in my experimental system?

  Rigorous validation should include:

  1. Molecular weight confirmation:

     • Western blot analysis should show a single band at approximately 51.6 kDa

     • Multiple bands may indicate degradation or non-specific binding

  2. Peptide competition assay:

     • Pre-incubate antibody with excess immunizing peptide

     • Compare staining with and without peptide competition

     • Specific signal should be blocked by the peptide

  3. Genetic approaches:

     • Test antibody on ENPP4 knockout/knockdown samples

     • Use overexpression systems as positive controls

     • Compare results with orthogonal detection methods

  4. Cross-reactivity assessment:

     • Test on tissues from different species to confirm expected reactivity

     • Evaluate potential cross-reactivity with other ENPP family members

     • Use bioinformatics to predict potential cross-reactive epitopes

• How can I effectively use ENPP4 antibodies in kidney development research?

  For studying ENPP4 in kidney development:

  1. Antibody selection:

     • Choose antibodies validated for developmental tissues

     • Consider using both N-terminal and C-terminal targeting antibodies to confirm results

  2. Staged developmental analysis:

     • Perform whole-mount immunohistochemistry on embryos at different developmental stages

     • Use antibodies specific for different nephron segments (e.g., 3G8 for proximal tubules, 4A6 for distal tubules) alongside ENPP4 staining

     • Analyze co-localization with markers such as slc5a1.1, slc12a1, clcnkb, and gata3

  3. Gain/loss of function approaches:

     • Use ENPP4 antibodies to confirm protein expression after mRNA injection or morpholino knockdown

     • Analyze phenotypes by immunostaining with pronephric tubule-specific antibodies

     • Quantify ectopic, enlarged, or reduced expression domains

  4. Double staining protocols:

     • For fluorescence: Use species-specific secondary antibodies with different fluorophores

     • For chromogenic detection: Sequential detection with different substrates (DAB and Vector Red)

     • Include proper controls for antibody cross-reactivity

• What techniques combine ENPP4 protein detection with functional enzymatic activity assessment?

  Integrating protein detection with activity measurement:

  1. In situ zymography combined with immunofluorescence:

     • Perform enzymatic activity assay using fluorogenic ATP substrates on tissue sections

     • Follow with immunofluorescence detection of ENPP4 protein

     • Co-localize activity with protein expression

  2. Cell-based functional assays:

     • Isolate ENPP4-expressing cells using antibody-based cell sorting

     • Measure enzymatic activity using biochemical assays

     • Correlate activity levels with protein expression quantified by flow cytometry or Western blot

  3. Proximity ligation assay:

     • Use ENPP4 antibodies with antibodies against potential substrates or interaction partners

     • Visualize protein-protein interactions in situ

     • Combine with functional ATP hydrolysis assays

  4. Antibody inhibition studies:

     • Use antibodies targeting different ENPP4 domains

     • Assess which antibodies inhibit enzymatic function

     • Map functional domains through antibody inhibition patterns