NEDD4L Antibody

What is NEDD4L and why is it an important research target?

NEDD4L (Neural precursor cell expressed developmentally down-regulated 4-like) is an E3 ubiquitin-protein ligase that accepts ubiquitin from E2 ubiquitin-conjugating enzymes and directly transfers it to targeted substrates. It plays crucial roles in multiple cellular pathways, making it an important research target . NEDD4L regulates ion channels, inhibits TGF-β signaling by triggering SMAD2 and TGFR1 ubiquitination and degradation, and promotes ubiquitination and internalization of various plasma membrane channels including ENaC, Nav1.2, Nav1.3, Nav1.5, Nav1.7, Nav1.8, Kv1.3, EAAT1, and CLC5 . Research has linked NEDD4L dysregulation to various diseases including cancer, cardiovascular disorders, and neurological conditions .

What are the common molecular weights observed for NEDD4L in research applications?

NEDD4L typically appears at molecular weights of 110-135 kDa in western blotting applications, though the exact weight may vary by cell type and experimental conditions . Specifically, antibodies have detected NEDD4L at:

• 110 kDa and 135 kDa bands (Cell Signaling Technology antibodies)

• 112 kDa calculated and 113 kDa observed (Assay Genie CAB8085)

• 112 kDa and 125 kDa observed (Proteintech 13690-1-AP)

• 130 kDa observed with calculated weight of 111,932 Da (Boster M01595)

These variations may reflect post-translational modifications, splice variants, or species-specific differences.

What species reactivity can be expected from commonly available NEDD4L antibodies?

Most commercial NEDD4L antibodies demonstrate cross-reactivity with multiple species, though some are species-specific . Common reactivity patterns include:

Researchers should validate antibody reactivity for their particular experimental system before proceeding with extended studies .

What are the recommended applications and dilutions for NEDD4L antibodies in different experimental techniques?

NEDD4L antibodies can be used across multiple experimental techniques with specific recommended dilutions :

Optimal dilutions should be determined empirically by each laboratory as they may vary depending on sample type, detection method, and specific research conditions .

How should samples be prepared for optimal NEDD4L detection in Western blotting?

For optimal NEDD4L detection in Western blotting, researchers should follow these methodological guidelines:

• Cell/tissue lysis: Use RIPA buffer or similar lysis buffers containing protease inhibitors to prevent protein degradation .

• Sample preparation: Heat samples at 95-100°C for 5 minutes in reducing sample buffer to denature proteins.

• Gel percentage: Use 8-10% SDS-PAGE gels due to NEDD4L's high molecular weight (110-135 kDa) .

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at lower voltage for longer periods (e.g., 30V overnight) to ensure efficient transfer of high molecular weight proteins.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST buffer for 1 hour at room temperature.

• Primary antibody incubation: Apply diluted antibody (e.g., 1:1000) in blocking buffer and incubate overnight at 4°C .

• Detection: Use appropriate secondary antibodies and ECL or other detection systems compatible with your imaging equipment.

Positive controls like PC-3, HT-1080, C6, or PC-12 cells have been validated for NEDD4L expression and can serve as experimental references .

What are the key considerations for immunohistochemical detection of NEDD4L?

For successful immunohistochemical detection of NEDD4L, researchers should consider:

• Fixation: 10% neutral buffered formalin is commonly used for tissue fixation before paraffin embedding.

• Antigen retrieval: Recommended methods include:

  • TE buffer pH 9.0 as the primary suggested method

  • Citrate buffer pH 6.0 as an alternative approach

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ and prevent non-specific binding with serum-based blocking solutions.

• Antibody dilution: Start with 1:50-1:200 dilution range for primary antibodies .

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal sensitivity.

• Detection systems: Use polymer-based detection systems for enhanced sensitivity and reduced background.

• Validated tissues: Human prostate cancer tissue and stomach cancer tissue have been validated for positive NEDD4L detection .

• Controls: Include both positive and negative controls in each IHC run to validate results.

Researchers should optimize these parameters for their specific experimental conditions and tissue types .

How can researchers distinguish between NEDD4 and NEDD4L (NEDD4-2) in their experiments?

Distinguishing between NEDD4 and NEDD4L requires careful antibody selection and experimental design:

What strategies can resolve common issues when working with NEDD4L antibodies in Western blotting?

When troubleshooting NEDD4L antibody issues in Western blotting, consider these methodological approaches:

• High background:

  • Increase blocking time or concentration (5-10% blocking agent)

  • Try alternative blocking agents (milk vs. BSA)

  • Increase washing frequency and duration (5-6 washes, 10 minutes each)

  • Reduce primary and secondary antibody concentrations

• Weak or no signal:

  • Increase protein loading (50-100 μg total protein)

  • Optimize transfer of high molecular weight proteins using lower voltage for longer time

  • Try fresher antibody preparations or different antibody clones

  • Use enhanced detection systems (high-sensitivity ECL)

  • Verify NEDD4L expression in your sample with validated positive controls like PC-3, HT-1080, C6, or PC-12 cells

• Multiple unexpected bands:

  • Use fresher sample preparations with additional protease inhibitors

  • Validate antibody specificity through knockout/knockdown controls

  • Consider post-translational modifications or splice variants

  • Try reducing agent concentration and sample heating conditions

  • Pre-absorb antibody with immunizing peptide where available

• Inconsistent results between experiments:

  • Standardize lysate preparation protocols

  • Implement loading controls

  • Prepare larger antibody aliquots to reduce freeze-thaw cycles

  • Maintain consistent incubation times and temperatures

How can phosphorylated forms of NEDD4L be specifically detected and what is their significance?

Phosphorylated forms of NEDD4L represent important regulatory states of the protein with distinct functional implications :

• Detection methods:

  • Use phospho-specific antibodies such as Phospho-NEDD4L (Ser448) Antibody

  • Employ phosphatase inhibitors in sample preparation buffers (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Utilize Phos-tag™ SDS-PAGE for mobility shift detection of phosphorylated forms

  • Perform phosphatase treatment controls to confirm specificity

• Key phosphorylation sites and kinases:

  • Ser342 and Ser448 are phosphorylated by AGC kinase family members including SGK1, Akt, and PKA

  • These phosphorylation events inhibit NEDD4L function as:
    a) Suppressor of ENaCs
    b) Regulator of TGF-β signaling through effects on Smad2/3 binding

• Experimental approaches for studying phosphorylation:

  • Kinase inhibitor treatments to block specific phosphorylation events

  • Site-directed mutagenesis (S→A) to create non-phosphorylatable mutants

  • Phosphomimetic mutations (S→D/E) to simulate constitutive phosphorylation

  • Co-immunoprecipitation studies to examine phosphorylation-dependent protein interactions

• Functional significance:

  • Phosphorylation at Ser342/Ser448 regulates the ubiquitin ligase activity of NEDD4L

  • These modifications modulate sodium channel trafficking and turnover

  • Changes in phosphorylation states may contribute to pathological conditions like hypertension through altered ENaC regulation

What disease models benefit from NEDD4L antibody research applications?

NEDD4L antibodies have proven valuable in studying multiple disease models:

• Hypertension and cardiovascular disorders:

  • NEDD4L regulates epithelial sodium channels (ENaCs), and mutations in ENaC PY motifs are associated with Liddle's syndrome, an autosomal dominant form of hypertension

  • Antibodies can track NEDD4L expression and phosphorylation changes in hypertensive models

  • Studies of NEDD4L's interaction with sodium channels inform potential therapeutic approaches

• Cancer research:

  • NEDD4L functions as both tumor suppressor and oncogene depending on cancer type

  • Antibodies enable analysis of NEDD4L expression in tumor tissues via IHC

  • Western blotting with NEDD4L antibodies helps quantify expression changes in cancer cell lines

  • Co-immunoprecipitation identifies cancer-specific interaction partners

• Neurological disorders:

  • NEDD4L regulates neuronal excitability through control of sodium channels (Nav1.2, Nav1.3, etc.)

  • Antibodies track NEDD4L distribution in brain tissue sections

  • Studies of NEDD4L in neurodegeneration models illuminate protein homeostasis mechanisms

• TGF-β signaling disorders:

  • NEDD4L negatively regulates TGF-β signaling by targeting Smad2 for degradation

  • Antibodies help investigate NEDD4L's role in fibrosis, wound healing, and embryonic development

Each research application benefits from specific methodological approaches using NEDD4L antibodies to track expression, localization, post-translational modifications, and protein-protein interactions.

How can researchers optimize co-immunoprecipitation experiments with NEDD4L antibodies?

Optimizing co-immunoprecipitation (Co-IP) with NEDD4L antibodies requires systematic attention to multiple methodological factors:

• Lysis buffer optimization:

  • Use mild non-denaturing buffers (e.g., 1% NP-40, 0.5% Triton X-100) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to maintain natural protein states

  • Adjust salt concentration (150-300 mM NaCl) to balance specificity and maintenance of interactions

• Antibody selection and preparation:

  • Choose antibodies validated for IP applications

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Crosslink antibodies to beads to prevent antibody contamination in eluates

• Experimental conditions:

  • Recommended antibody amounts: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Incubation conditions: 4°C overnight with gentle rotation

  • Washing stringency: Balance between maintaining specific interactions and reducing background

• Controls to include:

  • IgG control (same species as NEDD4L antibody)

  • Input sample (5-10% of starting material)

  • Reciprocal IP (if antibodies to interaction partners are available)

  • Sample treated with ubiquitination inhibitors to capture transient interactions

• Detection strategies:

  • Western blot with antibodies against expected interaction partners

  • Mass spectrometry for unbiased identification of interacting proteins

  • Proximity ligation assay as complementary method to validate interactions in situ

• Known interaction partners to investigate:

  • Sodium channels (NaVs and ENaCs)

  • TGF-β signaling components (Smad2/3, TGFR1)

  • Other ubiquitination targets (SGK1)

What are the emerging research applications for NEDD4L antibodies in cutting-edge methodologies?

NEDD4L antibodies are increasingly being incorporated into advanced research methodologies:

• Single-cell analysis approaches:

  • Imaging mass cytometry using metal-conjugated NEDD4L antibodies for tissue section profiling

  • Single-cell Western blotting to detect NEDD4L expression heterogeneity within populations

  • Multiplex immunofluorescence to examine NEDD4L alongside other pathway components

• Live-cell imaging applications:

  • Antibody-based biosensors to track NEDD4L localization during cellular processes

  • FRET/BRET approaches using antibody fragments to monitor NEDD4L interactions in real-time

  • Nanobody derivatives with greater tissue penetration for in vivo imaging

• Therapeutic development platforms:

  • Antibody-drug conjugates targeting cells with aberrant NEDD4L expression

  • Development of intrabodies to modulate NEDD4L function in specific cellular compartments

  • Proteolysis-targeting chimera (PROTAC) design guided by NEDD4L antibody epitope mapping

• Functional genomics integration:

  • Combined CRISPR screening with high-content antibody-based imaging

  • ChIP-seq using NEDD4L antibodies to identify genomic binding sites in transcriptional regulation

  • Spatial transcriptomics coupled with NEDD4L immunostaining to correlate expression patterns

• Structural biology applications:

  • Antibody-assisted cryo-EM to stabilize NEDD4L conformations

  • Epitope mapping to identify functional domains for targeted drug development

  • Selection of conformation-specific antibodies to distinguish active versus inactive NEDD4L states

These emerging methodologies expand the utility of NEDD4L antibodies beyond traditional applications and open new avenues for understanding NEDD4L biology in physiological and pathological contexts.

How should researchers design verification experiments to validate NEDD4L antibody specificity?

A systematic approach to validating NEDD4L antibody specificity should include multiple complementary methods:

• Genetic manipulation controls:

  • siRNA/shRNA knockdown of NEDD4L followed by Western blotting to demonstrate signal reduction

  • CRISPR/Cas9 knockout cell lines as negative controls

  • Overexpression systems using tagged NEDD4L constructs as positive controls

  • Testing across the six published knockdown/knockout systems cited in literature

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide where available

  • Titrate peptide concentrations to demonstrate dose-dependent signal reduction

  • Include irrelevant peptide controls to confirm specificity

• Cross-reactivity assessment:

  • Test antibody against purified recombinant NEDD4L and related family members

  • Evaluate specificity across species using samples from human, mouse, rat, and other relevant organisms

  • Compare reactivity patterns between multiple antibodies targeting different NEDD4L epitopes

• Technical validation:

  • Ensure appropriate positive controls (e.g., C6 cells, HT-1080 cells, PC-3 cells, PC-12 cells)

  • Compare results across different applications (WB, IP, IHC, IF) for consistency

  • Validate with orthogonal detection methods (e.g., mass spectrometry following immunoprecipitation)

• Documentation standards:

  • Record complete validation data including exposure times, sample amounts, and detection methods

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

  • Maintain consistent protocols between validation and experimental applications

What are the critical considerations when selecting between monoclonal and polyclonal NEDD4L antibodies?

The choice between monoclonal and polyclonal NEDD4L antibodies should be guided by experimental requirements:

The optimal choice depends on research goals:

• For detecting specific isoforms or phosphorylation states, monoclonal antibodies offer greater specificity

• For general detection of NEDD4L across applications, polyclonal antibodies typically provide higher sensitivity

• When studying interactions, consider epitope location to avoid interfering with binding sites

How do storage and handling conditions affect NEDD4L antibody performance over time?

Proper storage and handling are critical for maintaining NEDD4L antibody performance:

• Storage temperature recommendations:

  • Long-term storage: -20°C as recommended by most manufacturers

  • Short-term/frequent use: 4°C for up to one month

  • Avoid repeated freeze-thaw cycles that can degrade antibody performance

• Buffer composition effects:

  • Most NEDD4L antibodies are supplied in buffers containing:

    • PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

    • Some formulations include 0.1-0.5 mg/ml BSA as a stabilizer

  • Sodium azide prevents microbial contamination but can interfere with HRP activity in certain applications

• Aliquoting strategies:

  • Prepare single-use aliquots upon receipt to minimize freeze-thaw cycles

  • Maintain sterile conditions during aliquoting to prevent contamination

  • Document the date of aliquoting and number of freeze-thaw cycles

• Stability indicators:

  • Visible precipitation may indicate antibody denaturation

  • Progressive loss of signal intensity over time with consistent protocols

  • Increased background or non-specific binding

  • Manufacturers typically guarantee stability for one year after shipment when properly stored

• Reconstitution considerations:

  • Follow manufacturer-specific instructions for reconstitution volume

  • Allow antibodies to reach room temperature before opening to prevent condensation

  • Mix gently by inversion rather than vortexing to avoid protein denaturation

• Working solution handling:

  • Prepare fresh dilutions for each experiment when possible

  • For multi-day experiments, store diluted antibody at 4°C with preservatives

  • Avoid prolonged exposure to room temperature during experiments