NDRG1 Antibody

What is NDRG1 and why is it significant in scientific research?

NDRG1 is a highly conserved cytoplasmic protein (43 kDa) that plays crucial roles in cellular stress responses, hormone signaling, cell growth and differentiation. It belongs to the N-myc downstream regulated gene family and the alpha/beta hydrolase superfamily . NDRG1's significance stems from its multifaceted functions:

• Acts as both a tumor suppressor and potential oncogene depending on cellular context

• Regulates cell trafficking, particularly in Schwann cells

• Maintains peripheral nerve myelin sheath development

• Functions in p53/TP53-dependent mitotic spindle checkpoint

• Regulates microtubule dynamics and maintains euploidy

• Responds to environmental stressors including DNA damage, hypoxia, and heavy metals

The evolutionary conservation of NDRG1 across species highlights its fundamental biological significance .

What criteria should researchers consider when selecting an NDRG1 antibody for their experiments?

When selecting an NDRG1 antibody, researchers should consider:

• Target specificity: Choose antibodies that have been validated for specificity, such as those demonstrating low cross-reactivity with related proteins (e.g., antibodies showing <1% cross-reactivity with recombinant human N-myc)

• Species reactivity: Determine which species the antibody recognizes. Some antibodies detect NDRG1 across multiple species (human, mouse, rat, monkey), while others are species-specific

• Applications compatibility: Select antibodies validated for your specific application:

  • Western blotting (WB)

  • Immunoprecipitation (IP)

  • Immunofluorescence (IF)

  • Immunohistochemistry (IHC)

  • Flow cytometry (FC)

  • ELISA

• Antibody format: Consider whether you need unconjugated antibodies or those conjugated with specific tags (HRP, PE, FITC, Alexa Fluor conjugates)

• Clone type: Decide between monoclonal (greater specificity) or polyclonal (broader epitope recognition)

• Epitope location: Some antibodies target N-terminal regions, while others target C-terminal regions, which may detect different isoforms

How should researchers optimize Western blot protocols for NDRG1 detection?

Optimizing Western blot for NDRG1 detection requires attention to several key factors:

• Sample preparation:

  • Use RIPA buffer containing protease and phosphatase inhibitors

  • Determine protein content using the BCA method

  • Load approximately 40 μg of protein per lane

• Gel separation:

  • Use 10% SDS-PAGE for optimal resolution of NDRG1 (43-48 kDa)

• Transfer conditions:

  • Transfer to PVDF membrane for best results

  • Use appropriate transfer buffer depending on protein size

• Blocking:

  • Block with 5% milk in TBS with 0.05% Tween

  • For phospho-specific detection, use 5% BSA instead of milk

• Primary antibody incubation:

  • Recommended dilutions: 1:1000-1:5000 depending on antibody sensitivity

  • Incubate overnight at 4°C for optimal binding

• Detection considerations:

  • NDRG1 typically appears at 43-48 kDa

  • Multiple bands may be observed due to post-translational modifications or isoforms

  • Dp44mT treatment can result in multiple NDRG1 bands, suggesting upregulation of multiple isoforms

• Controls:

  • Human liver tissue and HeLa cells show consistent NDRG1 expression

  • NDRG1 knockout cells provide excellent negative controls

What are the best practices for immunofluorescence detection of NDRG1?

For optimal immunofluorescence detection of NDRG1:

• Cell preparation:

  • Culture cells on coverslips to appropriate density (1×10^4 cells/24 wells)

  • Consider testing both normoxic (21% O₂) and hypoxic (3.8% O₂) conditions as NDRG1 is hypoxia-responsive

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde

  • Wash with ice-cold 1× PBS

  • Permeabilize with 0.5% Triton-X for 5 minutes

• Blocking:

  • Block with 2% BSA in PBS for 30 minutes to reduce background

• Antibody incubation:

  • Dilute primary antibodies 1:20-1:500 depending on the specific antibody

  • For co-localization studies, create antibody cocktails (e.g., NDRG1 with Beta-catenin)

  • Incubate overnight in a humidified chamber

• Secondary antibody selection:

  • Choose appropriate fluorophore-conjugated secondary antibodies (Alexa Fluor 488, 594, etc.)

  • Alternatively, use directly conjugated primary antibodies like CoraLite®555-conjugated NDRG1 antibodies

• Visualization parameters:

  • For CoraLite®555-conjugated antibodies, use excitation/emission maxima wavelengths of 557 nm/570 nm

  • Counterstain nuclei with Hoechst 33342

• Control considerations:

  • Include untreated controls and NDRG1 knockout cells as negative controls

  • Dp44mT, mitoDFX, or mitoDFO treatment can be used as positive controls for NDRG1 upregulation

How can researchers investigate NDRG1's dual role as both tumor suppressor and oncogene?

NDRG1 exhibits context-dependent functions that require sophisticated experimental approaches:

• Cell line selection:

  • Use multiple cell lines from different cancer types

  • Compare results between cell lines where NDRG1 is reported as a tumor suppressor (colorectal, gastric cancers) versus potential oncogene (triple-negative breast cancer)

• Gene manipulation approaches:

  • Generate NDRG1 knockout cells using CRISPR-Cas9 targeting exon 4

  • Create cells overexpressing different NDRG1 isoforms:

    • Full-length (34,945)

    • N-terminally truncated isoforms (59,112 and 59,113)

• Functional assays:

  • Tumor growth: Xenograft models comparing WT, KO, and OE variants

  • Proliferation: Real-time monitoring assays (e.g., Incucyte)

  • Drug sensitivity: Test responses to therapies like cetuximab or iron chelators

  • Metastatic potential: Migration and invasion assays

• Molecular mechanism investigations:

  • Analyze EGFR expression and downstream signaling (RAS/RAF/ERK and PI3K/AKT/mTOR pathways)

  • Examine NDRG1 effects on endocytosis and degradation of EGFR induced by caveolin-1

  • Assess interaction with orphan Nur77 and effects on NF-κB transcriptional activity

• Subcellular localization studies:

  • Track NDRG1 distribution between membrane, cytoplasm, and nucleus

  • Correlate localization patterns with oncogenic or tumor-suppressive functions

Research has shown that full-length NDRG1 overexpression in MDA-MB-231 cells enhanced tumor growth, whereas NDRG1 knockout and overexpression of N-terminally truncated NDRG1 isoforms significantly reduced tumor growth . This highlights the complexity of NDRG1 function and the need for detailed isoform-specific studies.

What methodological approaches should be used to study NDRG1's role in drug resistance mechanisms?

To investigate NDRG1's involvement in drug resistance:

• Generating resistant cell models:

  • Develop drug-resistant cell lines through continuous exposure to increasing drug concentrations

  • Compare NDRG1 expression levels between parental and resistant cells

  • Analyze public databases like GEO (e.g., dataset GSE71210) for expression patterns in resistant vs. sensitive cells

• Gene modulation experiments:

  • Create NDRG1-overexpression and NDRG1-knockdown cells

  • Transfect with EGFR-overexpression plasmids or EGFR-siRNA to examine mechanism

  • Validate transfection efficiency via immunoblotting

• Drug sensitivity assays:

  • Determine IC₅₀ values using methods like CCK-8 assay

  • Compare results across modified cell lines:

    • NDRG1-overexpression cells: IC₅₀ for CTX in RKO cells (913.4 μg/ml)

    • NDRG1-overexpression cells with EGFR-overexpression: IC₅₀ (1559 μg/ml)

    • NDRG1-knockdown with EGFR-siRNA: IC₅₀ (957.4 μg/ml)

• Mechanistic pathway analysis:

  • Examine EGFR expression, phosphorylation, and membrane/cytoplasmic/nuclear distribution

  • Analyze downstream signaling pathways (RAS/RAF/ERK and PI3k/AKT/mTOR)

  • Investigate EGFR endocytosis and degradation mechanisms

• In vivo validation:

  • Use animal models to confirm mechanisms observed in vitro

  • Analyze patient-derived samples to correlate NDRG1 expression with treatment response

How can researchers effectively study the relationship between NDRG1 and cellular stress responses?

To investigate NDRG1's role in cellular stress responses:

• Stress induction methods:

  • Hypoxia: Culture cells at 3.8% O₂ in controlled O₂ incubators

  • Iron chelation: Treat with Dp44mT, mitoDFX, or mitoDFO at varying concentrations

  • DNA damage: UV irradiation or genotoxic agents

  • Heavy metal exposure: Nickel treatment

• Temporal dynamics analysis:

  • Monitor NDRG1 expression changes over time following stress induction

  • Analyze both mRNA (qPCR) and protein levels (Western blot)

  • Compare response kinetics across different stress types

• Subcellular localization tracking:

  • Use confocal immunofluorescence to track NDRG1 translocation

  • Perform cellular fractionation to quantify NDRG1 in different compartments

  • Correlate localization with specific stress responses

• Stress response pathway integration:

  • Examine relationship with HIF1α and HIF2α (hypoxia)

  • Analyze XBP1 interaction (ER stress)

  • Investigate p53/TP53 pathways (DNA damage response)

• Comparative analysis across cell types:

  • Compare responses in malignant (MCF7, MDA-MB-231) vs. non-malignant cells (MRC5)

  • Analyze tissue-specific differences in stress response (e.g., endothelial cells, fibroblasts)

For example, research has demonstrated that iron chelators increase NDRG1 expression in a dose-dependent manner, with non-targeted chelator Dp44mT inducing stronger responses than mitochondrially targeted chelators (mitoDFX and mitoDFO) .

How should researchers interpret multiple NDRG1 bands on Western blots?

Multiple NDRG1 bands on Western blots require careful interpretation:

• Expected band patterns:

  • Primary NDRG1 band: 43 kDa (calculated molecular weight)

  • Secondary bands: 46-48 kDa (post-translationally modified forms)

• Potential causes of multiple bands:

  • Isoform detection: Human NDRG1 has multiple isoforms, including full-length (34,945) and N-terminally truncated variants (59,112 and 59,113)

  • Post-translational modifications: Phosphorylation by SGK1 (at Thr328, Ser330, Thr346, Thr356, and Thr366) and subsequent phosphorylation by GSK-3

  • Stress-induced modifications: Treatment with Dp44mT results in multiple NDRG1 bands, suggesting stress-responsive modifications

  • Proteolytic processing: Partial degradation during sample preparation

• Validation approaches:

  • Use NDRG1 knockout cells as negative controls

  • Compare results with different NDRG1 antibodies targeting different epitopes

  • Treat samples with phosphatases to determine if bands are due to phosphorylation

  • Use isoform-specific overexpression as positive controls

• Application-specific considerations:

  • For quantification, determine whether to measure a specific band or the sum of all bands

  • For mechanistic studies, consider which isoforms/modifications are most relevant to your research question

What are common challenges in NDRG1 antibody experiments and how can they be overcome?

Researchers face several challenges when working with NDRG1 antibodies:

• Background signal in immunostaining:

  • Problem: High background in immunofluorescence or IHC

  • Solution: Optimize blocking (use 2-5% BSA), increase washing steps, titrate antibody dilution (1:50-1:500), and consider antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Variable detection across tissues:

  • Problem: Inconsistent detection in different tissue types

  • Solution: Adjust fixation protocols based on tissue type, optimize antibody concentration for each tissue, and consider tissue-specific expression levels (NDRG1 is highly expressed in placenta, prostate, kidney, small intestine, and ovary)

• Isoform-specific detection:

  • Problem: Difficulty distinguishing between NDRG1 isoforms

  • Solution: Use antibodies targeting different epitopes (N-terminal vs. C-terminal), compare with overexpression controls of specific isoforms, and use higher resolution gels (12-15%) for better separation

• Cross-reactivity concerns:

  • Problem: Potential cross-reactivity with related proteins

  • Solution: Validate specificity using NDRG1 knockout cells, perform direct ELISAs to confirm minimal cross-reactivity (<1% with related proteins like N-myc), and compare results with multiple antibodies

• Phosphorylation-specific detection:

  • Problem: Difficulty distinguishing phosphorylated forms

  • Solution: Use phospho-specific antibodies if available, include phosphatase-treated controls, and optimize gel conditions for phospho-protein detection

How can researchers reconcile conflicting data regarding NDRG1's role in cancer progression?

To address contradictory findings about NDRG1's function in cancer:

• Context-dependent analysis:

  • Approach: Systematically compare NDRG1 function across multiple cancer types and cellular contexts

  • Implementation: Use identical methodologies across different cell types to enable direct comparison

• Isoform-specific investigations:

  • Approach: Distinguish between effects of different NDRG1 isoforms

  • Implementation: Create cells expressing specific isoforms (full-length vs. N-terminally truncated) and compare their functional effects

• Subcellular localization correlations:

  • Approach: Correlate NDRG1's function with its subcellular distribution

  • Implementation: Combine subcellular fractionation with functional assays to determine if localization patterns explain functional differences

• Pathway integration analysis:

  • Approach: Examine how NDRG1 interacts with different signaling networks in different contexts

  • Implementation: Analyze NDRG1 interaction partners using immunoprecipitation followed by mass spectrometry, and correlate with functional outcomes

• Prognostic correlation studies:

  • Approach: Correlate NDRG1 expression patterns with clinical outcomes

  • Implementation: Analyze expression and subcellular localization in patient samples and correlate with disease progression, treatment response, and survival

Research has demonstrated that NDRG1 can function as either a tumor suppressor or oncogene depending on the cellular context. In breast cancer, full-length NDRG1 overexpression enhanced tumor growth, while N-terminally truncated isoforms reduced growth . Additionally, NDRG1 enhances cetuximab sensitivity in colorectal cancer by suppressing EGFR expression , highlighting its complex, context-dependent roles.

How can NDRG1 be leveraged as a biomarker in cancer diagnosis and treatment stratification?

Leveraging NDRG1 as a biomarker requires systematic approaches:

• Biomarker validation methodology:

  • Conduct retrospective analysis correlating NDRG1 expression with treatment outcomes

  • Develop standardized detection protocols for clinical samples

  • Establish expression thresholds that correlate with therapeutic responses

• Treatment response prediction:

  • Use NDRG1 expression to predict cetuximab sensitivity in colorectal cancer

  • Analyze NDRG1 expression patterns before and after treatment

  • Correlate subcellular localization with treatment efficacy

• Multi-marker panel development:

  • Integrate NDRG1 with other markers for improved predictive power

  • Combine with markers of metabolic status (given NDRG1's association with metabolic gene expression programs)

  • Incorporate with hypoxia markers (since NDRG1 is hypoxia-responsive)

• Technical considerations for clinical implementation:

  • Develop reproducible IHC protocols suitable for pathology laboratories

  • Standardize scoring systems for NDRG1 expression and localization

  • Validate across multiple patient cohorts

Research has shown that NDRG1 expression and subcellular localization are clinically relevant biomarkers for poor prognosis in breast cancer, suggesting potential utility in treatment stratification .

What are the methodological considerations for studying NDRG1 in vascular biology and inflammation?

To investigate NDRG1 in vascular biology:

• Endothelial cell model selection:

  • Use primary human endothelial cells (HUVECs, HAECs) for physiological relevance

  • Consider tissue-specific endothelial cells for organ-specific studies

  • Compare results between normal and disease-associated endothelial cells

• Inflammation induction protocols:

  • Stimulate cells with specific cytokines (IL-1β, TNF-α) to model inflammation

  • Document concentration-dependent and time-dependent effects

  • Compare acute vs. chronic inflammatory conditions

• Loss-of-function approaches:

  • Use NDRG1 short hairpin RNA for knockdown studies

  • Generate endothelial cell-specific NDRG1 knockout mice for in vivo studies

  • Validate knockdown efficiency before proceeding with experiments

• Functional readouts:

  • Measure expression of inflammatory markers (cytokines/chemokines, adhesion molecules)

  • Assess procoagulant molecules (PAI-1, TF) and antithrombotic factors (TM, t-PA)

  • Evaluate transcriptional activity of Nur77, NF-κB, and AP-1

• In vivo models:

  • Use carotid artery ligation to study neointima formation

  • Employ atherosclerosis models in endothelial cell-specific NDRG1 knockout mice

  • Assess arterial thrombosis models

Research has shown that NDRG1 knockdown attenuates IL-1β and TNF-α-induced expression of inflammatory molecules and reduces procoagulant molecule expression, suggesting NDRG1 is essential for endothelial inflammation .

How should researchers design experiments to investigate NDRG1's role in cellular metabolism and lipid handling?

For studying NDRG1's metabolic functions:

• Metabolic profiling approaches:

  • Compare metabolic patterns between NDRG1 knockout and wildtype cells

  • Use metabolomics to identify affected pathways

  • Focus on lipid metabolism given NDRG1's suggested role in lipid handling

• Lipid trafficking assays:

  • Employ fluorescently labeled lipids to track trafficking

  • Use subcellular fractionation to determine lipid distribution

  • Compare normal vs. stressed conditions (e.g., lipid loading)

• Integration with metabolic stress responses:

  • Study NDRG1 responses to metabolic stressors (glucose deprivation, hypoxia)

  • Examine relationship with metabolic sensors (AMPK, mTOR)

  • Investigate connections to HIF and hypoxia-induced metabolic adaptations

• Cancer metabolism connections:

  • Correlate NDRG1 expression with 18-fluorodeoxyglucose uptake in tumors

  • Investigate NDRG1's role in Warburg-like metabolic gene expression

  • Study NDRG1's function in regulating lipid fate in cancer cells

• Therapeutic targeting considerations:

  • Test metabolic pathway inhibitors in NDRG1-modified cells

  • Evaluate iron chelators' effects on metabolism in relation to NDRG1 induction

  • Explore combined targeting of NDRG1 and metabolic pathways

Evidence suggests NDRG1 performs an important pro-survival function in regulating the fate of lipids in breast cancer cells and is expressed in a Warburg-like metabolic gene expression program common to many solid tumors .