ENTPD4 Antibody

Basic Research Questions

• What is ENTPD4 and why is it significant in research?

ENTPD4 is a member of the GDA1/CD39 NTPase protein family that catalyzes the hydrolysis of nucleoside triphosphates and diphosphates in a calcium- or magnesium-dependent manner, with a preference for pyrimidine nucleotides. In humans, the canonical protein has a reported length of 616 amino acid residues and a molecular mass of 70.3 kDa . ENTPD4's subcellular localization is primarily in the Golgi apparatus, lysosomes, and cytoplasmic vesicles, making it an important marker for these compartments . The protein exists in at least two isoforms with distinct functional properties, is ubiquitously expressed across many tissue types, and undergoes post-translational modifications including glycosylation . ENTPD4 plays crucial roles in nucleotide metabolism and has emerging significance in immune regulation, cancer biology, and inflammatory responses.

• What are the primary applications for ENTPD4 antibodies in research?

ENTPD4 antibodies are utilized across multiple research applications, with specific requirements for each technique:

Western blot remains the most commonly validated application across different manufacturer antibodies, with consistent detection of ENTPD4 at approximately 70 kDa . The wide range of recommended dilutions reflects variations in antibody affinity, format, and sample types, requiring optimization for specific experimental conditions.

• What criteria should guide ENTPD4 antibody selection for different research applications?

Selecting the appropriate ENTPD4 antibody requires consideration of multiple factors:

Different antibody formats have distinct advantages and limitations for ENTPD4 detection:

For challenging experimental designs, researchers should consider using multiple antibodies targeting different ENTPD4 epitopes to validate results .

• What are the optimal sample preparation methods for detecting ENTPD4 in different sample types?

Sample preparation significantly impacts ENTPD4 detection across different experimental contexts:

For Western blot analysis:

• Cell lysates should be prepared in RIPA buffer containing protease inhibitors to prevent degradation

• Heat samples at 95°C for 5 minutes in reducing buffer containing SDS and DTT

• Load 25-50 μg of total protein per lane for cell lysates, 10-20 μg for tissue extracts

• Jurkat cells, human placenta tissue, mouse heart, and rat testis serve as excellent positive controls

For immunohistochemistry:

• Fixation in 4% paraformaldehyde (24 hours at 4°C) followed by paraffin embedding preserves antigenicity

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes significantly improves detection

• For frozen sections, fixation in cold acetone (-20°C for 10 minutes) maintains epitope accessibility

• Blocking with 5% normal serum matching the secondary antibody species reduces background

For immunofluorescence:

• Fix cells in 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (10 minutes) for intracellular epitopes

• For membrane-associated epitopes, use milder detergents like 0.1% saponin

• Counter-stain with organelle markers to confirm subcellular localization

Optimizing these parameters for your specific antibody and sample type is essential for reliable ENTPD4 detection.

• How can I validate the specificity of ENTPD4 antibodies?

Rigorous validation of ENTPD4 antibodies ensures reliable and reproducible results:

• Positive controls: Use tissues or cell lines with known ENTPD4 expression, such as Jurkat cells, human placenta tissue, mouse heart, and rat testis

• Western blot analysis: Verify detection at the expected molecular weight (approximately 70 kDa)

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide (e.g., the 40-69 amino acid sequence for N-terminal antibodies) to demonstrate specificity

• Knockdown/knockout verification: Compare antibody reactivity in wild-type versus ENTPD4 siRNA-treated or knockout samples

• Multiple antibody approach: Use antibodies targeting different ENTPD4 epitopes to confirm expression patterns

• Recombinant protein: Test reactivity against purified recombinant ENTPD4 protein (available commercially with various tags)

• Orthogonal methods: Correlate protein detection with mRNA expression data from RT-PCR or RNA-seq

Implementation of multiple validation approaches strengthens confidence in experimental findings and helps resolve potential discrepancies between different antibodies.

Advanced Research Questions

• How should I troubleshoot non-specific binding or inconsistent results with ENTPD4 antibodies?

Non-specific binding and inconsistent results are common challenges when working with ENTPD4 antibodies:

A systematic optimization approach for ENTPD4 Western blots includes:

• Test multiple primary antibody dilutions (starting with manufacturer recommendations)

• Compare different blocking agents (5% milk vs. 5% BSA) for optimal signal-to-noise ratio

• Extend wash steps (5 × 5 minutes with TBST)

• Verify sample integrity with positive controls (Jurkat cells, human placenta tissue)

• Consider deglycosylation treatment to address potential glycosylation-induced heterogeneity

For each optimization step, maintain careful records of conditions and results to establish the most reliable protocol for your specific experimental system.

• How do post-translational modifications affect ENTPD4 antibody binding and experimental design?

ENTPD4 undergoes post-translational modifications, primarily glycosylation, which significantly impact antibody detection:

• Effects on antibody binding:

  • Glycosylation can mask epitopes, particularly in the extracellular/luminal domains

  • Different glycosylation patterns across cell types and tissues may affect detection sensitivity

  • The apparent molecular weight of ENTPD4 may vary from the calculated 70.3 kDa due to glycosylation

• Methodological strategies:

  • Enzymatic deglycosylation: Treat samples with PNGase F or Endo H prior to SDS-PAGE

  • Protocol: Denature protein samples, add 500 units of PNGase F, incubate for 1-2 hours at 37°C

  • Compare treated and untreated samples to assess the impact of glycosylation

  • Select antibodies targeting regions less likely to be modified (cytoplasmic domains)

  • Use antibodies raised against non-glycosylated recombinant proteins for consistent detection

• Experimental considerations:

  • Include both deglycosylated and native samples to comprehensively analyze ENTPD4

  • Compare detection patterns across different tissues with known glycosylation variations

  • Document shifts in molecular weight following deglycosylation treatments

  • Consider how glycosylation might affect functional assays when using antibodies

Awareness of these modification-related issues is essential for accurate interpretation of ENTPD4 expression data and proper experimental design.

• What approaches can resolve contradictory results between different ENTPD4 antibodies?

When different ENTPD4 antibodies yield contradictory results, a systematic analytical approach is necessary:

• Epitope mapping analysis:

  • Map the exact target regions of each antibody (e.g., N-terminal region AA 40-69 vs. central region AA 350-470 )

  • Consider epitope accessibility in different sample preparation methods

  • Evaluate potential for epitope masking by protein interactions or modifications

• Isoform-specific detection:

  • ENTPD4 has two reported isoforms with distinct functional properties:

    • Isoform 1: Preferentially hydrolyzes UTP and TTP

    • Isoform 2: Has broader substrate specificity

  • Determine if your antibodies recognize one or both isoforms

  • Correlate with isoform-specific mRNA expression data

• Orthogonal validation approaches:

  • Use recombinant ENTPD4 protein to test each antibody's reactivity

  • Employ RNA interference to confirm specificity (observe corresponding decrease in signal)

  • Combine immunoprecipitation with mass spectrometry to verify protein identity

  • Correlate protein detection with enzymatic activity assays

• Comprehensive protocol optimization:

  • Systematically test each antibody across multiple sample preparation methods

  • Optimize individual conditions for each antibody (fixation, antigen retrieval, dilution)

  • Document all variables that affect detection sensitivity and specificity

By implementing this systematic approach, researchers can determine which antibody or combination of antibodies provides the most accurate representation of ENTPD4 biology in their experimental system.

• How can I optimize ENTPD4 antibody protocols for immunohistochemistry in challenging tissue samples?

Optimizing ENTPD4 detection in challenging tissues requires careful consideration of fixation and antigen retrieval methods:

A systematic optimization workflow for challenging tissues includes:

• Fixation comparison:

  • Test multiple fixation methods (4% PFA, methanol, acetone, zinc-based fixatives)

  • Optimize fixation duration (4, 12, 24, 48 hours) to balance preservation and antigenicity

  • For archival FFPE tissues, extend antigen retrieval times (20-40 minutes)

• Antigen retrieval optimization:

  • Compare heat-induced methods:

    • Citrate buffer (10 mM, pH 6.0)

    • Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 9.0)

    • Glycine-HCl (0.1 M, pH 3.0)

  • Test enzymatic methods:

    • Proteinase K (20 μg/mL, 10-15 minutes)

    • Pepsin (0.05%, 5-10 minutes)

  • Optimize retrieval duration (10, 20, 30 minutes)

• Signal amplification systems:

  • Compare direct detection vs. biotin-streptavidin systems

  • Test tyramide signal amplification for low-abundance detection

  • Evaluate polymer-based detection systems

This methodical approach allows optimization of ENTPD4 detection even in challenging tissue samples while maintaining specificity and minimizing background.

• What strategies can be used to study ENTPD4 function using antibodies beyond simple detection?

ENTPD4 antibodies can be leveraged for functional studies beyond basic expression analysis:

• Functional blocking studies:

  • Identify antibodies targeting enzymatic domains that can inhibit activity

  • Test inhibition of nucleotidase activity using in vitro assays with recombinant ENTPD4

  • Measure substrate depletion and product formation (UTP → UDP → UMP)

  • Compare with small molecule inhibitors to validate effects

• Protein-protein interaction analysis:

  • Immunoprecipitation with ENTPD4 antibodies followed by mass spectrometry

  • Protocol: Lyse cells in non-denaturing buffer, incubate with ENTPD4 antibody (5 μg), capture with Protein A/G beads, wash extensively, elute and analyze interacting proteins

  • Proximity ligation assays to visualize and quantify interactions in situ

  • FRET studies using fluorescently-labeled antibody fragments

• Trafficking and localization dynamics:

  • Live-cell imaging with non-perturbing antibody fragments

  • Pulse-chase experiments with antibodies against extracellular epitopes

  • Co-localization studies with markers for subcellular compartments:

    • Golgi (GM130, TGN46)

    • Lysosomes (LAMP1, LAMP2)

    • Endosomes (EEA1, Rab5, Rab7)

• Structure-function relationships:

  • Mapping accessible epitopes under different conditions

  • Monitoring conformational changes using conformation-sensitive antibodies

  • Correlating structural features with enzymatic activity

These advanced applications enable researchers to move beyond descriptive studies to mechanistic investigations of ENTPD4 biology and function.

• How can I use ENTPD4 antibodies to investigate its role in disease models?

ENTPD4 antibodies can be powerful tools for investigating disease relevance in various models:

• Cancer research applications:

  • Analyze ENTPD4 expression across tumor tissues and paired normal samples

  • Correlate expression with patient outcomes and clinicopathological features

  • Examine subcellular distribution changes in malignant transformation

  • Study correlation with purinergic signaling alterations in the tumor microenvironment

• Inflammatory and immune disorders:

  • Quantify ENTPD4 expression changes during inflammatory responses

  • Compare expression in immune cell subsets under normal and pathological conditions

  • Investigate relationship with extracellular nucleotide levels in inflammatory microenvironments

  • Correlate with disease severity in tissue samples from patients with inflammatory disorders

• Methodological approaches:

  • Tissue microarray analysis for high-throughput screening across multiple patient samples

  • Multi-color immunofluorescence to assess co-expression with disease markers

  • Laser capture microdissection combined with Western blot for region-specific analysis

  • Quantitative image analysis for objective assessment of expression changes

• Therapeutic target assessment:

  • Blocking antibody studies to assess functional relevance in disease models

  • Monitor expression changes in response to treatments

  • Develop antibody-guided drug delivery approaches

By combining these approaches, researchers can establish ENTPD4's role in disease pathogenesis and evaluate its potential as a diagnostic marker or therapeutic target.

• What controls should be included when using ENTPD4 antibodies for critical research publications?

Rigorous controls are essential for ensuring reproducibility and validity in ENTPD4 research:

Implementation strategies for different experimental contexts:

• For Western blot:

  • Include recombinant ENTPD4 protein as positive control

  • Run molecular weight markers to confirm expected size (70 kDa)

  • Test multiple antibodies against different epitopes when possible

  • Include loading controls (β-actin, GAPDH) for normalization

• For immunohistochemistry/immunofluorescence:

  • Process positive and negative control tissues alongside experimental samples

  • Include secondary antibody-only control sections

  • Use blocking peptide competition on serial sections

  • Perform parallel staining with multiple validated antibodies

• For high-impact publications:

  • Combine multiple detection methods (WB, IHC, IF)

  • Validate with orthogonal approaches (mRNA quantification)

  • Document all antibody information (catalog number, lot, dilution)

  • Provide quantitative analysis with appropriate statistical methods

Implementing these comprehensive controls significantly strengthens the reliability of ENTPD4 research findings and enhances reproducibility across different laboratories.

• What are the key considerations when using ENTPD4 antibodies for co-localization studies?

Co-localization studies require specific methodological considerations to ensure accurate results:

• Antibody compatibility:

  • Use primary antibodies from different host species to avoid cross-reactivity

  • If using same-species antibodies, employ sequential immunostaining protocols

  • Verify that antibodies do not compete for overlapping epitopes

  • Test for cross-reactivity between secondary antibodies

• Microscopy optimization:

  • Collect images below the diffraction limit to minimize false co-localization

  • Use confocal microscopy with appropriate pinhole settings

  • Correct for chromatic aberration with specific calibration tools

  • Employ super-resolution techniques for detailed subcellular localization

• Quantitative analysis:

  • Calculate Pearson's or Manders' correlation coefficients for objective assessment

  • Employ object-based co-localization for discrete structures

  • Use appropriate thresholding methods consistently across samples

  • Analyze multiple fields and cells for statistical validity

• Biological controls:

  • Include known co-localizing and non-co-localizing pairs as controls

  • For ENTPD4, appropriate controls include:

    • Co-localization with Golgi markers (positive control)

    • Co-localization with mitochondrial markers (negative control)

  • Use fluorescently-tagged ENTPD4 as complementary approach

By implementing these methodological considerations, researchers can generate reliable co-localization data regarding ENTPD4's subcellular distribution and protein-protein interactions.

• How can ENTPD4 antibodies be used to explore nucleotide metabolism and purinergic signaling?

ENTPD4's enzymatic activity directly impacts nucleotide metabolism and purinergic signaling pathways:

• Enzymatic activity correlation:

  • Immunoprecipitate ENTPD4 using specific antibodies and measure enzymatic activity

  • Protocol: Capture ENTPD4 with antibody-coated beads, measure nucleotide hydrolysis rates using HPLC or colorimetric assays

  • Correlate protein levels (by Western blot) with enzymatic activity in different tissues/cells

  • Use blocking antibodies to inhibit ENTPD4 function and assess metabolic consequences

• Signaling pathway analysis:

  • Examine co-localization with purinergic receptors (P2X, P2Y) using dual immunofluorescence

  • Investigate ENTPD4 expression changes after purinergic receptor stimulation

  • Assess calcium signaling alterations after modulating ENTPD4 expression/activity

  • Map complete purinergic signaling networks using antibodies against multiple components

• Metabolic impact assessment:

  • Correlate ENTPD4 expression with extracellular nucleotide profiles

  • Compare wild-type and ENTPD4-depleted cells for changes in nucleotide metabolism

  • Integrate with metabolomics analysis to comprehensively assess nucleotide pathways

  • Study tissue-specific metabolic consequences in normal and pathological states

These approaches can elucidate ENTPD4's specific contributions to nucleotide homeostasis and purinergic signaling networks in various physiological and pathological contexts.

• What future directions are emerging for ENTPD4 antibody research applications?

Emerging technologies and applications are expanding the utility of ENTPD4 antibodies in research:

• Advanced imaging applications:

  • Super-resolution microscopy for nanoscale localization

  • Expansion microscopy to physically enlarge samples for improved resolution

  • Lattice light-sheet microscopy for rapid 3D imaging with minimal photodamage

  • Correlative light and electron microscopy for ultrastructural context

• Single-cell analysis:

  • Mass cytometry (CyTOF) with metal-conjugated ENTPD4 antibodies

  • Single-cell Western blotting for heterogeneity assessment

  • Imaging mass cytometry for tissue spatial analysis at single-cell resolution

  • Integration with single-cell transcriptomics for multi-omics analysis

• Therapeutic development:

  • Antibody-drug conjugates targeting ENTPD4-expressing cells

  • Bispecific antibodies linking ENTPD4 to immune effector cells

  • Development of function-modulating antibodies as therapeutic agents

  • Imaging agents for visualization of ENTPD4 expression in vivo

• Emerging disease connections:

  • Recent studies suggest potential roles for ENTPD4 in:

    • Immune regulation and inflammatory disorders

    • Cancer metabolism and purinergic signaling

    • Neurodegenerative diseases

    • Metabolic disorders

  • Antibodies will be essential tools for exploring these emerging connections

These innovative approaches demonstrate the continuing evolution of ENTPD4 antibody applications in basic research, translational studies, and potential therapeutic development.