NDRG1 Antibody

What is NDRG1 and why is it important in cellular research?

NDRG1 (N-myc downstream-regulated gene 1) is a 43 kDa protein belonging to the NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. It is ubiquitously expressed but shows particularly high expression in nonproliferating, differentiating tissues. NDRG1 is crucial for cellular homeostasis and responds to environmental changes through its involvement in cellular stress responses and hormone signaling pathways . The protein contains an NDRG domain (amino acids 286-316) and three tandem 10 amino acid hydrophilic repeats (amino acids 339-368), with the human version consisting of 394 amino acids in total . Its evolutionary conservation across species (human NDRG1 shows 95% amino acid identity to mouse NDRG1 over amino acids 11-267) highlights its fundamental biological significance .

What experimental applications are NDRG1 antibodies typically used for?

NDRG1 antibodies are versatile research tools employed in multiple experimental techniques including:

• Western blotting (WB) for protein expression quantification

• Immunoprecipitation (IP) for protein-protein interaction studies

• Immunofluorescence (IF) for subcellular localization analysis

• Immunohistochemistry (IHC) for tissue expression patterns

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

For optimal results, researchers should select antibodies validated specifically for their intended application. For instance, the NDRG1 Antibody (B-5) has been validated for all the above applications with mouse, rat, and human samples . Western blot detection typically reveals NDRG1 at approximately 43 kDa, as demonstrated with human liver tissue and HeLa cell lysates .

What are the key differences between monoclonal and polyclonal NDRG1 antibodies?

Monoclonal antibodies like NDRG1 Antibody (B-5) offer high specificity to a single epitope and consistent lot-to-lot reproducibility. They are produced from a single B-cell clone, resulting in identical antibodies that recognize the same epitope of NDRG1 . This makes them excellent for applications requiring precise epitope targeting.

Polyclonal antibodies, such as Goat Anti-Human NDRG1 Antigen Affinity-purified Polyclonal Antibody, recognize multiple epitopes on the NDRG1 protein. They are typically produced by immunizing animals (often goats or rabbits) with recombinant NDRG1 protein fragments, like the E. coli-derived recombinant human NDRG1 (Met111-Asp267) used for the antibody in search result . Polyclonal antibodies often provide stronger signals due to multiple epitope binding but may show more batch-to-batch variation.

The choice between monoclonal and polyclonal depends on the specific research needs—monoclonal for consistent epitope recognition across experiments, polyclonal for enhanced signal detection.

How should researchers optimize western blot protocols for NDRG1 detection?

For optimal western blot detection of NDRG1:

• Sample preparation: Use reducing conditions as demonstrated in scientific data from R&D Systems, which successfully detected NDRG1 at approximately 43 kDa .

• Antibody concentration: Start with 1 μg/mL for primary antibody incubation based on validated protocols (e.g., with Goat Anti-Human NDRG1 Antigen Affinity-purified Polyclonal Antibody) .

• Membrane selection: PVDF membranes have been successfully used for NDRG1 detection.

• Buffer system optimization: Use appropriate immunoblot buffer groups; for example, Immunoblot Buffer Group 8 was used successfully in the R&D Systems protocol .

• Secondary antibody selection: Match to your primary antibody species—for example, HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019) for goat primary antibodies .

• Positive controls: Include HeLa cell lysates or human liver tissue lysates, which reliably express detectable NDRG1 levels .

• Molecular weight verification: Always confirm detection at the expected 43 kDa size to distinguish from potential isoform variants or non-specific binding.

What factors affect the specificity of NDRG1 detection in immunological assays?

Several factors can influence NDRG1 antibody specificity:

• Cross-reactivity with NDRG family members: Test for potential cross-reactivity with related proteins. For example, direct ELISA testing showed less than 1% cross-reactivity with recombinant human N-myc when using the Human NDRG1 Antibody (AF5209) .

• Isoform recognition: Consider the three potential isoform variants of NDRG1—two involving alternate start sites at Met82 and Met286, and a third showing amino acid substitutions in the first 34 residues . Antibodies may differ in their ability to detect these variants.

• Epitope accessibility: Post-translational modifications or protein-protein interactions may mask antibody binding sites. This is particularly relevant as NDRG1 interacts with orphan Nur77 (nuclear receptor) in endothelial cells .

• Fixation effects: For immunohistochemistry or immunofluorescence, fixation methods can alter epitope availability. Establish optimal fixation protocols for your specific NDRG1 antibody.

• Tissue-specific expression patterns: NDRG1 is predominantly expressed in placenta, prostate, kidney, small intestine, and ovary , which should be considered when choosing positive and negative control tissues.

How does NDRG1 contribute to vascular inflammation and what methods best capture this role?

NDRG1 plays a significant role in vascular inflammation and endothelial activation. Research has demonstrated that:

• Expression pattern: NDRG1 expression is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions .

• Functional significance: Knockdown of NDRG1 using lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β and TNF-α-induced expression of cytokines/chemokines and adhesion molecules .

• Antithrombotic effects: Inhibition of NDRG1 significantly attenuates the expression of procoagulant molecules such as PAI-1 and TF, while increasing the expression of thrombomodulin and t-PA, exerting potent antithrombotic effects in endothelial cells .

Recommended methodological approaches:

• Loss-of-function studies: Use NDRG1 short hairpin RNA as demonstrated in cardiovascular research .

• Endothelial cell-specific knockout models: Generate endothelial cell-specific NDRG1 knockout mice to study in vivo effects on neointima formation, atherosclerosis, and arterial thrombosis .

• Protein-protein interaction studies: Investigate NDRG1 interactions with Nur77 and effects on NF-κB using co-immunoprecipitation and transcriptional reporter assays .

• Signaling pathway analysis: Examine NDRG1's effects on cytokine-induced MAPK activation, c-Jun phosphorylation, and AP-1 transcriptional activity .

What role does NDRG1 play in cancer therapy response and how can researchers study this function?

NDRG1 has emerged as a significant modulator of cancer therapy response, particularly in relation to EGFR-targeted therapies:

• Cetuximab sensitivity enhancement: Research shows that NDRG1 enhances the sensitivity of colorectal cancer cells to cetuximab (CTX), the first monoclonal antibody targeting EGFR .

• Mechanisms of action: NDRG1 inhibits EGFR expression, blocks EGFR phosphorylation, and reduces EGFR distribution in the cell membrane, cytoplasm, and nucleus .

• Downstream pathway effects: NDRG1 suppresses EGFR downstream signaling through the RAS/RAF/ERK and PI3K/AKT/mTOR pathways .

• Endocytosis regulation: NDRG1 attenuates the endocytosis and degradation of EGFR induced by caveolin-1 (Cav1) .

Recommended methodological approaches:

• Protein expression and phosphorylation analysis: Western blotting to assess EGFR expression levels and phosphorylation status in response to NDRG1 modulation .

• Subcellular fractionation: Determine EGFR distribution in membrane, cytoplasmic, and nuclear fractions with and without NDRG1 expression .

• Signaling pathway assessment: Analyze activation of RAS/RAF/ERK and PI3K/AKT/mTOR pathways using phospho-specific antibodies .

• Drug sensitivity assays: Measure cell viability, apoptosis, and proliferation in response to cetuximab treatment under conditions of NDRG1 overexpression or knockdown .

• Animal models: Validate findings in xenograft models to assess NDRG1's effect on tumor response to cetuximab in vivo .

How should researchers address contradictory findings about NDRG1 function across different experimental systems?

Contradictory findings about NDRG1 function may arise due to context-dependent roles of this protein. For example, while NDRG1 was identified as highly upregulated in anergic B cells, knockout studies revealed it to be functionally redundant for lymphocyte anergy . To address such contradictions:

• Consider cell type specificity: NDRG1 functions may vary significantly between cell types. For instance, its role in endothelial cells during inflammation differs from its function in lymphocytes during anergy induction .

• Evaluate signal context: NDRG1 is upregulated by B cell receptor activation (signal one) but suppressed by co-stimulation (signal two) , suggesting its regulation and function are highly context-dependent.

• Assess compensatory mechanisms: The finding that NDRG1 is dispensable for B cell tolerance despite being highly upregulated suggests functional redundancy . Researchers should investigate potential compensatory pathways.

• Use multiple model systems: Test hypotheses in both in vitro cellular systems and in vivo animal models, as demonstrated in studies of NDRG1's role in cetuximab sensitivity .

• Employ complementary approaches: Combine genetic manipulation (knockout/knockdown) with pharmacological approaches to distinguish between developmental compensation and acute functional requirements.

• Control for isoform variation: The three potential NDRG1 isoform variants may have distinct functions, so researchers should specify which isoform(s) they are studying.

What are the best approaches for studying NDRG1 protein-protein interactions?

To effectively study NDRG1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Use NDRG1 antibodies suitable for immunoprecipitation, such as NDRG1 Antibody (B-5) . Co-IP has successfully demonstrated NDRG1's interaction with orphan Nur77 (nuclear receptor) in endothelial cells .

• Conjugated antibody options: Consider using NDRG1 Antibody (B-5) AC (agarose conjugated) for pull-down assays, which provides 500 μg/ml concentration in 25% agarose .

• Proximity ligation assay (PLA): This technique can visualize protein interactions in situ with high specificity and sensitivity, providing spatial information about NDRG1 interactions.

• Yeast two-hybrid screening: For unbiased discovery of novel NDRG1 interaction partners.

• Domain mapping: Focus on the NDRG domain (aa 286-316) and the three tandem hydrophilic repeats (aa 339-368) as potential protein interaction regions .

• Mass spectrometry: After immunoprecipitation, use mass spectrometry to identify interacting proteins in an unbiased manner.

• Functional validation: Confirm biological relevance of identified interactions through functional assays, such as transcriptional reporter assays used to demonstrate NDRG1's inhibition of Nur77 and NF-κB transcriptional activity .

How can researchers effectively investigate NDRG1's role in the regulation of EGFR signaling and cancer therapy resistance?

Building on findings that NDRG1 enhances cetuximab sensitivity by modulating EGFR signaling , researchers should consider:

• Comprehensive pathway analysis: Investigate NDRG1's effects on all branches of EGFR signaling, including STAT pathways alongside the already-established RAS/RAF/ERK and PI3K/AKT/mTOR effects .

• Receptor trafficking studies: Use fluorescently tagged EGFR to track its internalization, recycling, and degradation in real-time imaging with and without NDRG1 expression.

• Structural biology approaches: Determine whether NDRG1 directly interacts with EGFR or affects its conformation, potentially altering antibody binding or kinase activity.

• Resistance mechanism screening: Employ CRISPR-Cas9 screens to identify genes that modify NDRG1's effect on cetuximab sensitivity, potentially uncovering new resistance mechanisms.

• Combination therapy exploration: Test whether NDRG1 modulation can enhance sensitivity to other EGFR-targeted therapies or overcome acquired resistance.

• Biomarker development: Evaluate NDRG1 expression as a predictive biomarker for cetuximab response in patient samples using immunohistochemistry with validated NDRG1 antibodies .

• Therapeutic targeting: Investigate whether small molecules or peptides can mimic NDRG1's beneficial effects on EGFR signaling as a potential therapeutic strategy.

What experimental designs best elucidate the apparently redundant role of NDRG1 in lymphocyte anergy despite its strong upregulation?

The finding that Ndrg1 is significantly upregulated in anergic B cells compared to naïve B cells, yet dispensable for lymphocyte anergy , presents an intriguing research question. To investigate this paradox:

• Temporal expression analysis: Use time-course experiments with NDRG1 antibodies to precisely track when NDRG1 is upregulated during anergy induction and whether its expression correlates with specific phases of the process.

• Compensation identification: Employ RNA-sequencing of wild-type versus NDRG1-deficient anergic cells to identify upregulated genes that might compensate for NDRG1's absence.

• NDRG family redundancy: Investigate whether other NDRG family members are upregulated in NDRG1-deficient anergic cells, potentially explaining functional redundancy.

• Acute versus chronic deletion: Compare acute NDRG1 depletion (using inducible systems) with constitutive knockout to distinguish between immediate requirements and developmental compensation.

• Context-dependent functions: Test whether NDRG1's dispensability is specific to certain lymphocyte subsets or activation conditions by varying antigen dose, affinity, and costimulatory signals.

• Stress response integration: Investigate whether NDRG1 becomes essential for anergy under specific stress conditions, given its known role in stress responses .

• Systems biology approach: Use network analysis to position NDRG1 within the broader transcriptional network of anergy to understand its connections and potential redundancies.