dennd10 Antibody

Basic Research Questions

• What is DENND10 and why is it significant for cancer research?

DENND10 is an endosomal protein that functions as an intrinsic regulator of cell migration through modifying the tumor microenvironment via autocrine EV release. Research has demonstrated that DENND10 expression is significantly associated with poor prognosis in multiple cancer types and is upregulated in metastatic breast cancer cell lines . Studies using knockout models have shown that DENND10 deletion leads to defective EV biogenesis due to impaired endolysosomal trafficking, resulting in reduced cell spreading, migration, invasion, and metastatic potential in vivo . These findings position DENND10 as a potential therapeutic target for tumor metastasis intervention.

• What types of DENND10 antibodies are available for research applications?

Currently, researchers can access several types of DENND10 antibodies:

The antibodies are typically available in liquid form in PBS containing glycerol, BSA, and preservatives, requiring storage at -20°C to maintain efficacy .

• How should DENND10 antibodies be validated before experimental use?

Proper validation of DENND10 antibodies should include:

• Western blot analysis using positive control lysates from cells known to express DENND10 and negative controls (DENND10 knockout cells)

• Testing for cross-reactivity across multiple species when relevant (human/mouse reactivity is common for commercially available antibodies)

• Determination of optimal antibody concentration through titration experiments (typically 1:500-2000 dilution range for Western blot applications)

• Verification of subcellular localization pattern (DENND10 is primarily localized to late endosomes)

• Confirming specificity by immunoprecipitation followed by mass spectrometry

These validation steps are crucial as inappropriate antibody specificity can lead to misleading experimental outcomes and irreproducible results.

• What are the recommended protocols for using DENND10 antibodies in Western blot applications?

For optimal Western blot results with DENND10 antibodies:

• Sample preparation: Lyse cells in RIPA buffer containing protease inhibitors

• Protein separation: Load 20-40 μg of protein per lane on 10% SDS-PAGE gels

• Transfer: Transfer proteins to PVDF membrane at 100V for 1-2 hours

• Blocking: Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute DENND10 antibody 1:500-2000 in blocking buffer and incubate overnight at 4°C

• Washing: Wash membrane 3× with TBST for 10 minutes each

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

• Detection: Visualize using enhanced chemiluminescence reagents

Expected result: DENND10 should appear as a band at approximately 45-50 kDa. Comparing results with DENND10 knockout cell lysates can confirm antibody specificity .

Advanced Research Questions

• How can researchers effectively use DENND10 antibodies to investigate its role in extracellular vesicle biogenesis?

Investigating DENND10's role in EV biogenesis requires a multi-faceted approach:

• Comparative analysis: Generate DENND10 knockout cell lines using CRISPR/Cas9 gene editing (as demonstrated with 4T1 breast cancer cells)

• EV isolation protocol:

  • Collect conditioned medium from wild-type and DENND10-KO cells

  • Remove cellular debris by centrifugation at 2000×g for 20 min

  • Filter through 0.22 μm filters

  • Ultracentrifuge at 100,000×g for 70 min to pellet EVs

  • Wash EVs in PBS and repeat ultracentrifugation

• Analysis methods:

  • Nanoparticle tracking analysis to quantify EV size distribution and concentration

  • Western blot using DENND10 antibodies to confirm presence in wild-type EVs and absence in DENND10-KO EVs

  • Transmission electron microscopy to examine EV morphology

  • Proteomic analysis to identify altered cargo composition

Research has shown that DENND10 knockout leads to defective EV biogenesis, with changes in both quantity and composition of EVs, particularly affecting extracellular matrix (ECM) and adhesion molecules .

• What are the current challenges in using DENND10 antibodies for immunofluorescence studies of endolysosomal trafficking?

Immunofluorescence studies of DENND10 in endolysosomal trafficking face several technical challenges:

• Fixation method selection: DENND10's endosomal localization requires careful fixation to preserve membrane structure. Paraformaldehyde (4%) with 0.1% glutaraldehyde often provides better preservation than methanol fixation.

• Permeabilization optimization: Excessive permeabilization can disrupt endosomal membranes, while insufficient permeabilization limits antibody access. A titration of detergent concentrations (0.1-0.5% Triton X-100 or 0.05-0.1% saponin) should be tested.

• Co-localization markers: For accurate endolysosomal tracking, co-staining with established markers is essential:

  • Early endosomes: EEA1, Rab5

  • Late endosomes: Rab7, M6PR

  • Lysosomes: LAMP1, Cathepsin D

• Signal-to-noise ratio: Background fluorescence can obscure the specific DENND10 signal. Strategies to improve this include:

  • Extended blocking (2+ hours with 5% BSA)

  • Higher antibody dilutions with longer incubation times

  • Use of highly cross-adsorbed secondary antibodies

• Live-cell imaging limitations: Current antibodies are primarily suitable for fixed-cell applications, limiting real-time trafficking studies.

Research has shown that DENND10 knockout cells exhibit delayed trafficking to lysosomes, with accumulated immature precursors of Cathepsin D and elevated levels of LAMP1 and M6PR, indicating compensatory lysosomal biogenesis .

• How can researchers interpret contradictory DENND10 antibody staining patterns in different cancer cell lines?

Contradictory DENND10 antibody staining patterns across different cancer cell lines may stem from several factors that require systematic investigation:

• Expression level variation: Quantify baseline DENND10 expression using qRT-PCR and Western blot across cell lines to establish whether differences are due to actual expression variations.

• Alternative splicing: Design primers to detect potential isoforms that might lack specific epitopes recognized by the antibody. RNA-seq analysis can identify cancer-specific splice variants.

• Post-translational modifications: Investigate whether DENND10 undergoes differential phosphorylation, ubiquitination, or other modifications in different cancer contexts using:

  • Phosphatase treatment before Western blotting

  • Immunoprecipitation followed by mass spectrometry

  • Phospho-specific antibodies if available

• Protein-protein interactions: Different binding partners may mask antibody epitopes. Co-immunoprecipitation studies can identify cancer-specific interaction partners.

• Subcellular redistribution: Cancer cells often display altered vesicular trafficking. Compare DENND10 localization with endosomal/lysosomal markers across cell lines using confocal microscopy.

• Control experiments for validation:

  • DENND10 knockout cells as negative controls

  • Antibody pre-absorption with recombinant protein

  • Testing multiple antibodies targeting different epitopes

When analyzing staining patterns, researchers should consider that DENND10's role in endolysosomal trafficking suggests its distribution may naturally vary depending on the metabolic and migratory state of different cancer cells .

• What methodological approaches can be used to study the relationship between DENND10 and the extracellular matrix using antibody-based techniques?

Studying DENND10's relationship with the extracellular matrix (ECM) requires integrating multiple antibody-based approaches:

• Comparative proteomics of EVs:

  • Isolate EVs from wild-type and DENND10-KO cells

  • Perform quantitative mass spectrometry to identify differential ECM components

  • Validate findings using Western blot with specific antibodies against identified ECM proteins

• Immunofluorescence co-localization:

  • Co-stain for DENND10 and ECM components (fibronectin, collagens, laminins)

  • Analyze using super-resolution microscopy to detect potential interactions

  • Quantify Pearson's correlation coefficients to measure association strength

• Proximity ligation assay (PLA):

  • Use DENND10 antibody paired with antibodies against ECM proteins

  • PLA signal indicates proteins are within 40 nm of each other

  • Quantify interaction signals in different cellular compartments

• Secretome analysis:

  • Immunoprecipitate DENND10 from conditioned medium

  • Identify co-precipitating ECM proteins by mass spectrometry

  • Validate interactions with reverse co-immunoprecipitation

• Functional rescue experiments:

  • Add purified ECM proteins to DENND10-KO cells

  • Assess restoration of spreading and migration defects

  • Compare effectiveness of different ECM components

Research has demonstrated that DENND10 knockout results in a distinct EV compositional landscape with remodeled profiles of ECM and adhesion molecules. Importantly, exogenous application of ECM molecules rescued the spreading and migration defects of DENND10-KO cells, confirming the functional relationship between DENND10 and the ECM in regulating cell motility .

• What are the optimal methods for using DENND10 antibodies in immunoprecipitation experiments to identify novel interaction partners?

For effective immunoprecipitation (IP) of DENND10 to identify novel interaction partners:

• Antibody selection and validation:

  • Test multiple DENND10 antibodies for IP efficiency

  • Validate specificity using DENND10-KO cells as negative controls

  • Consider epitope location relative to potential protein interaction domains

• Lysis buffer optimization:

  • For membrane-associated interactions: Use gentler detergents (0.5-1% NP-40, 0.5% CHAPS)

  • For stronger interactions: RIPA buffer

  • Include protease/phosphatase inhibitors and maintain cold temperature

• IP protocol refinement:

  • Pre-clear lysates with protein A/G beads to reduce background

  • Optimize antibody-to-lysate ratio (typically 2-5 μg antibody per 1 mg protein)

  • Consider crosslinking antibody to beads to prevent antibody contamination in mass spectrometry

  • Include appropriate controls (IgG control, DENND10-KO cells)

• Interaction validation workflow:

  Technique	Application	Advantage
  Mass spectrometry	Unbiased identification	Discovers novel interactions
  Reverse IP	Validation	Confirms bidirectional interaction
  Proximity ligation assay	In situ confirmation	Visualizes interaction in cellular context
  FRET/BRET	Live cell dynamics	Measures interaction kinetics
  GST pull-down	Domain mapping	Identifies specific binding regions

• Specialized approaches for endosomal interactions:

  • Subcellular fractionation before IP to enrich endosomal compartments

  • BioID or APEX2 proximity labeling to capture transient interactions

  • Vesicle immunoprecipitation using anti-DENND10 to isolate intact vesicles

Studies indicate that DENND10 regulates endolysosomal trafficking, suggesting it likely interacts with Rab GTPases and other trafficking machinery components .

• How can researchers design experiments to investigate the differential effects of DENND10 in metastasis across multiple cancer types using antibody-based approaches?

Designing comprehensive experiments to investigate DENND10's role in metastasis across cancer types requires:

• Expression profiling across cancer types:

  • Tissue microarray immunohistochemistry using validated DENND10 antibodies

  • Correlation of expression with clinical outcome data

  • Quantitative analysis of staining intensity and subcellular localization

• Multi-cancer cell line panel analysis:

  • Western blot quantification of DENND10 across diverse cancer cell lines

  • Correlation with established invasion/migration capabilities

  • Generation of DENND10-KO in representative lines from each cancer type

• Comparative functional assays:

  Assay Type	Measurement	Relevance to Metastasis
  Transwell migration	Cell motility	Early metastatic step
  Matrigel invasion	ECM penetration	Invasiveness
  3D spheroid invasion	Collective migration	Tumor microenvironment interaction
  Adhesion assays	Cell-matrix attachment	Metastatic niche establishment
  In vivo metastasis models	Organ-specific metastasis	Physiological relevance

• Cancer-specific EV characterization:

  • Isolate EVs from multiple DENND10-KO cancer cell types

  • Compare proteomic profiles to identify common vs. cancer-specific cargoes

  • Test functional effects of these EVs on recipient cells

• Mechanistic pathway investigation:

  • Phospho-specific antibody arrays to identify differential signaling

  • Co-immunoprecipitation followed by cancer-specific interactome analysis

  • Rescue experiments with wild-type vs. mutant DENND10

Research has shown that DENND10 expression is significantly associated with poor prognosis across multiple cancer types, with a notably important role in breast cancer metastasis . Bioinformatics data mining can help identify the cancer types most likely to be dependent on DENND10-mediated metastasis mechanisms for targeted investigation.

• What advanced computational approaches can be used to improve anti-DENND10 antibody design for research applications?

Advanced computational approaches for improved anti-DENND10 antibody design include:

• Epitope prediction and optimization:

  • Use structural prediction tools like AlphaFold and IgFold to model DENND10 tertiary structure

  • Identify surface-exposed, conserved epitopes with high antigenicity

  • Select epitopes distant from functional domains to minimize interference with protein activity

• Machine learning for antibody sequence optimization:

  • Apply deep learning models trained on massive antibody sequence datasets to predict binding properties

  • Implement RFdiffusion approaches to design antibodies with specified framework structures

  • Utilize complementarity determining region (CDR) optimization for improved specificity

• Molecular dynamics simulations:

  • Simulate antibody-DENND10 interactions to predict binding stability

  • Identify potential conformational changes upon binding

  • Optimize binding kinetics through iterative design improvements

• High-throughput virtual screening:

  • Generate in silico libraries of potential antibody candidates

  • Computationally dock candidates to modeled DENND10 structure

  • Rank candidates based on binding energy calculations

• Developability prediction:

  • Calculate physiochemical properties to predict antibody solubility and stability

  • Identify potential post-translational modification sites that might affect function

  • Predict immunogenicity profiles for potential in vivo applications

Similar approaches have been successfully applied to developing broadly neutralizing antibodies against dengue virus, demonstrating that computational methods and machine learning can accelerate antibody discovery . The IgFold method has shown particular promise, predicting antibody structures with similar or better quality than alternative methods in significantly less time (under one minute) .

• How can researchers systematically troubleshoot inconsistent results when using DENND10 antibodies across different experimental platforms?

Systematic troubleshooting of inconsistent DENND10 antibody results requires a structured approach:

• Antibody validation matrix:
  Create a comprehensive validation checklist for each experimental platform:

  Platform	Positive Control	Negative Control	Expected Signal	Troubleshooting Variables
  Western Blot	Recombinant DENND10	DENND10-KO lysate	45-50 kDa band	Blocking agent, incubation time, antibody dilution
  Immunofluorescence	Cells with confirmed expression	DENND10-KO cells	Endosomal pattern	Fixation method, permeabilization, antibody concentration
  Immunohistochemistry	Known positive tissue	Antibody pre-absorption	Cell-type specific	Antigen retrieval, detection system, section thickness
  Flow Cytometry	Transfected cells	Isotype control	Specific population shift	Permeabilization protocol, compensation settings

• Epitope accessibility analysis:

  • Map the antibody epitope relative to protein domains and post-translational modification sites

  • Test multiple antibodies targeting different regions of DENND10

  • Consider native versus denatured conditions affecting epitope exposure

• Sample preparation variables:

  • Systematic comparison of lysis buffers (RIPA, NP-40, Triton X-100)

  • Test effect of various protease/phosphatase inhibitor combinations

  • Evaluate impact of freeze-thaw cycles on epitope integrity

• Technical standardization:

  • Implement absolute quantification using purified standards

  • Establish inter-laboratory validation protocols

  • Use automated systems where possible to reduce technical variability

• Root cause analysis workflow:

  1. Document all experimental conditions precisely

  2. Change only one variable at a time

  3. Include appropriate controls with each experiment

  4. Maintain a centralized database of all results for pattern identification

  5. Consult antibody manufacturer regarding lot-to-lot variation