NDRG1 Antibody, FITC conjugated

What is NDRG1 and what are its key biological functions?

NDRG1 is a 43-kDa stress-responsive protein involved in multiple cellular processes including hormone responses, cell growth, and differentiation. It acts as a tumor suppressor in many cell types and plays a significant role in p53/TP53-mediated caspase activation and apoptosis, though it is necessary but not sufficient for these processes . NDRG1 has important functions in cell trafficking, particularly in Schwann cells, and is essential for the maintenance and development of the peripheral nerve myelin sheath. The protein is required for vesicular recycling of CDH1 and TF, and may function in lipid trafficking .

NDRG1 protects cells from spindle disruption damage, functions in p53/TP53-dependent mitotic spindle checkpoint pathways, regulates microtubule dynamics, and helps maintain euploidy . Recent studies have also demonstrated NDRG1's involvement in regulating classical signaling pathways such as Ras/Raf/ERK and PI3K/Akt/mTOR, which are downstream targets of EGFR . Additionally, NDRG1 plays a critical role in endothelial inflammation, thrombotic responses, and vascular biology .

What applications are FITC-conjugated NDRG1 antibodies best suited for?

FITC-conjugated NDRG1 antibodies are primarily optimized for flow cytometry applications, allowing direct detection without secondary antibodies . This direct conjugation simplifies experimental protocols, reduces background, and enables multiparameter analysis. The FITC Anti-NDRG1 (phospho T346) antibody, for example, is specifically validated for flow cytometry with human samples .

Beyond flow cytometry, these antibodies can be valuable for immunofluorescence microscopy when studying NDRG1's subcellular localization, particularly in research examining its roles in cell trafficking, epithelial barrier integrity, and vascular inflammation . The green fluorescence of FITC (excitation ~493nm, emission ~519nm) makes it compatible with standard fluorescence microscopy filter sets and confocal systems .

For phosphorylation-specific variants like the phospho T346 antibody, these conjugates enable researchers to track specific activation states of NDRG1 in response to various stimuli or in different disease contexts .

How do FITC-conjugated NDRG1 antibodies compare with other fluorophore conjugations?

When selecting a fluorophore for NDRG1 detection, researchers should consider spectral properties, photostability, and experimental requirements. FITC has an excitation maximum at ~493nm and emission at ~519nm, producing green fluorescence that is compatible with standard filter sets .

Alternative conjugates available for NDRG1 antibodies include:

• AF350: Excitation 346nm/Emission 442nm (blue)

• AF405: Excitation 401nm/Emission 421nm (blue-violet)

• AF488: Excitation 493nm/Emission 519nm (green, similar to FITC but more photostable)

• AF555: Excitation 555nm/Emission 565nm (yellow-green)

• AF594: Excitation 591nm/Emission 614nm (red)

• AF647: Excitation 651nm/Emission 667nm (far-red)

• AF680: Excitation 679nm/Emission 702nm (near-infrared)

• AF750: Excitation 749nm/Emission 775nm (near-infrared)

For tissues with high autofluorescence in the green spectrum, far-red conjugates like AF647 may provide better signal-to-noise ratios. For multiparameter experiments, selecting complementary fluorophores with minimal spectral overlap is essential. AF488 offers similar spectral properties to FITC but with enhanced brightness and photostability, making it advantageous for extended imaging sessions or samples with low NDRG1 expression .

What controls are essential when studying NDRG1 phosphorylation in cellular stress responses?

When investigating NDRG1 phosphorylation dynamics, particularly at sites like T346, implementing appropriate controls is critical for reliable results:

Essential biological controls:

• Total NDRG1 detection alongside phospho-specific detection to normalize for expression changes

• Time-matched untreated samples to establish baseline phosphorylation levels

• Positive control treatments known to induce specific phosphorylation sites (e.g., SGK1 activators for T346 phosphorylation)

• NDRG1 knockdown/knockout samples as negative controls for antibody specificity verification

Technical validation controls:

• Phosphatase-treated samples to confirm phospho-specificity

• Phospho-blocking peptide competition to verify epitope specificity

• Isotype-matched FITC-conjugated control antibodies at equivalent concentrations

• Fluorescence minus one (FMO) controls for flow cytometry experiments

Pathway validation controls:

• Specific kinase inhibitors targeting enzymes known to phosphorylate NDRG1

• Upstream pathway modulators to confirm expected signaling cascades

• Phosphorylation site mutants (e.g., T346A) as definitive negative controls

These controls help distinguish genuine phosphorylation changes from technical artifacts and enable confident attribution of observed changes to specific cellular conditions rather than non-specific effects or antibody cross-reactivity.

How can researchers optimize protocols for detecting low-level NDRG1 expression?

Detecting low-abundance NDRG1 expression can be challenging with standard protocols. Consider these methodological optimizations:

Signal amplification strategies:

• Extended primary antibody incubation (overnight at 4°C)

• Sequential antibody labeling using biotinylated secondary antibodies followed by streptavidin-FITC

• Tyramide signal amplification (TSA) for up to 100-fold signal enhancement

Flow cytometry optimization:

• Systematic PMT voltage optimization to maximize signal-to-noise ratio

• Reduced flow rate to increase signal integration time

• Consideration of brighter fluorophores like PE or APC if FITC signal remains insufficient

Microscopy enhancements:

• Confocal microscopy with optimized pinhole settings

• Increased numerical aperture objectives

• Deconvolution algorithms to improve signal-to-noise ratio

• Anti-fade mounting media to prevent photobleaching during extended imaging

Sample preparation refinements:

• Optimized gentle fixation to preserve antigenic epitopes

• Tested permeabilization protocols to enhance antibody access

• Extended blocking steps to reduce background fluorescence

• Cell enrichment techniques if NDRG1 expression is restricted to specific subpopulations

These approaches can significantly improve detection sensitivity while maintaining specificity, allowing for reliable analysis of NDRG1 expression in challenging experimental contexts.

What are the optimal fixation and permeabilization methods for NDRG1 detection?

The choice of fixation and permeabilization methods significantly impacts NDRG1 detection, particularly for phosphorylated forms:

Recommended fixation protocols:

• For total NDRG1: 4% paraformaldehyde (15-20 minutes at room temperature) preserves protein localization while maintaining cell morphology

• For phospho-NDRG1 epitopes: Methanol fixation (-20°C for 10 minutes) often better preserves phosphoepitopes like T346

• For some applications, a combination approach using PFA followed by methanol permeabilization may provide optimal results

Permeabilization considerations:

• For flow cytometry: Commercial intracellular staining kits optimized for phospho-proteins generally provide superior results

• For microscopy: 0.1-0.5% Triton X-100 (5-10 minutes) for nuclear and cytoplasmic NDRG1 detection

• For membrane-associated NDRG1 pools: Gentler permeabilization with 0.1% saponin may better preserve membrane associations

Special considerations:

• Include phosphatase inhibitors in all buffers when studying phosphorylated NDRG1

• Process samples rapidly to preserve phosphorylation states

• Standardize fixation timing across experimental conditions, as extended fixation can reduce antibody binding efficiency

Preliminary experiments comparing different protocols are strongly recommended, as the optimal method may vary depending on the specific epitope being detected and the experimental system being used.

How can FITC-conjugated phospho-specific NDRG1 antibodies be used to study signaling pathways?

Phosphorylation-specific antibodies such as FITC Anti-NDRG1 (phospho T346) enable sophisticated investigation of NDRG1's role in signaling networks:

Time-course analysis approaches:

• Treat cells with relevant stimuli for varying durations

• Process for flow cytometry using FITC-conjugated phospho-NDRG1 antibodies

• Quantify phosphorylation dynamics to understand temporal regulation

• Correlate with activation of known upstream kinases and downstream effectors

Inhibitor studies:

• Pretreat cells with specific kinase inhibitors before stimulation

• Measure impact on NDRG1 phosphorylation by flow cytometry

• This approach can delineate the specific kinases responsible for NDRG1 phosphorylation in different contexts

• Particularly relevant for exploring NDRG1's roles in Ras/Raf/ERK and PI3K/Akt/mTOR pathways

Multiparameter signaling analysis:

• Combine FITC-phospho-NDRG1 detection with other signaling markers

• The AHA study demonstrated NDRG1's impact on MAPK activation, c-Jun phosphorylation, and AP-1 transcriptional activity

• Flow cytometric analysis using FITC-NDRG1 with phospho-specific antibodies for these signaling proteins enables correlation of NDRG1 status with pathway activation

These approaches allow researchers to position NDRG1 within complex signaling networks and understand how its phosphorylation affects downstream cellular processes in contexts like tumor suppression, endothelial inflammation, and epithelial barrier regulation.

What methodological approaches are effective for studying NDRG1's role in endothelial inflammation?

The AHA Journals article reveals NDRG1's critical role in endothelial inflammation and thrombotic responses. To study this function:

Endothelial cell models and stimulation protocols:

• Primary human umbilical vein endothelial cells (HUVECs) treated with pro-inflammatory cytokines (IL-1β, TNF-α)

• The study showed NDRG1 knockdown markedly attenuated both TNF-α and IL-1β-induced expression of VCAM-1 and ICAM-1

• Time-course experiments tracking NDRG1 expression during inflammatory activation

• Application of fluid shear stress using flow chambers to mimic physiological conditions, as NDRG1 expression is regulated by shear stress

Functional assays correlated with NDRG1 detection:

• Monocyte adhesion assays combined with NDRG1 expression analysis

• The study demonstrated NDRG1 knockdown suppressed IL-1β-induced adhesion of U937 cells to HUVECs by approximately 70%

• Measuring NDRG1 expression by flow cytometry and correlating with adhesion molecule expression provides mechanistic insights

Molecular interaction studies:

• NDRG1 was shown to interact with orphan nuclear receptor Nur77

• The DNA-binding domain (DBD) of Nur77 was specifically responsible for binding NDRG1

• NDRG1 overexpression dose-dependently inhibited Nur77 response element-driven luciferase activity

• These findings suggest NDRG1 regulates endothelial inflammation partly through modulating Nur77's transcriptional activity

In vivo experimental approaches:

• Endothelial cell-specific NDRG1 knockout mice showed markedly attenuated neointima and atherosclerosis formation

• These models provide systems for testing how NDRG1 modulation affects inflammatory vascular diseases

These methodologies enable comprehensive investigation of NDRG1's role in vascular inflammation, potentially leading to new therapeutic strategies for inflammatory vascular diseases.

How does NDRG1 expression influence response to EGFR-targeted therapies like cetuximab?

The Nature article highlights NDRG1's significant impact on sensitivity to cetuximab (CTX), an EGFR-targeted therapy:

Expression correlation studies:

• NDRG1 was found to enhance the sensitivity of colorectal cancer to CTX

• Analysis of public GEO dataset GSE71210 showed NDRG1 was overexpressed in CTX-resistant cells (1.89-fold increase)

Mechanistic investigation approaches:

• The study revealed NDRG1 affects EGFR expression, distribution, phosphorylation, endocytosis, and degradation

• NDRG1-overexpression cells became insensitive to CTX after EGFR-overexpression plasmid transfection

• NDRG1 could promote Cav1 ubiquitylation in colorectal cancer cells, potentially affecting EGFR endocytosis

• These findings suggest NDRG1 modulates CTX sensitivity through regulating EGFR trafficking and signaling

Experimental validation methodology:

• EGFR-overexpression plasmids and EGFR-siRNA transfection in NDRG1-modified cells

• Drug sensitivity testing by CCK-8 assay

• Western blot verification of modulation efficiency

• These approaches helped establish the causal relationship between NDRG1, EGFR levels, and drug response

These findings suggest NDRG1 expression levels could potentially serve as a biomarker for predicting response to EGFR-targeted therapies. Flow cytometric analysis using FITC-conjugated NDRG1 antibodies could provide a quantitative method for assessing this predictive biomarker in patient samples.

How can researchers study NDRG1's role in epithelial barrier integrity?

Research highlighted in the Wiley Online Library article demonstrates NDRG1's importance in maintaining airway epithelial barrier integrity:

Barrier function assays correlated with NDRG1 expression:

• Transepithelial electrical resistance (TEER) measurements

• Dextran permeability assays

• The study showed NDRG1 knockdown decreased TEER and increased dextran permeability

• Immunocytochemistry revealed NDRG1 knockdown disrupted tight junctions

Mechanistic investigation approaches:

• Analysis of tight junction molecule expression after NDRG1 knockdown

• The study found NDRG1 knockdown specifically decreased claudin-9 expression

• This effect was selective, as other claudin family molecules, E-cadherin, and ZO proteins were unaffected

• Subsequent claudin-9 knockdown experiments confirmed its importance for barrier function

Tissue expression pattern analysis:

• Immunohistochemical analysis in patient samples

• Strong NDRG1 expression was observed in ciliated epithelial cells in nasal tissues from chronic rhinosinusitis patients

• Low NDRG1 expression was noted in goblet cells and damaged epithelial cells

• These patterns suggest cell type-specific roles for NDRG1 in airway epithelium

Gene expression profiling during barrier development:

• The study identified NDRG1 as a gene induced during epithelial cell barrier development

• Similar approaches could be used to study NDRG1's role in barrier formation in other epithelial tissues

These methodologies provide a framework for investigating NDRG1's role in maintaining epithelial barriers, with potential relevance to respiratory diseases, inflammatory conditions, and wound healing processes.

What considerations are important when incorporating FITC-conjugated NDRG1 antibodies into multiparameter flow cytometry panels?

Multiparameter flow cytometry allows simultaneous analysis of NDRG1 expression alongside other cellular markers. Key considerations include:

Panel design strategies:

• FITC emits in the green spectrum (~519 nm), requiring selection of complementary fluorophores with minimal spectral overlap

• Common compatible fluorophores include PE (yellow), APC (red), and Pacific Blue (blue)

• When studying cells with high autofluorescence, consider brighter fluorophores like AF488 instead of FITC

Control requirements:

• Single-color controls for each fluorochrome are essential for compensation

• Fluorescence Minus One (FMO) controls that include all fluorochromes except FITC-NDRG1 help determine proper gating

• Isotype controls conjugated to FITC establish background fluorescence levels

Intracellular staining optimization:

• Commercial kits designed for intracellular phospho-protein detection often provide superior results

• When combining with surface marker detection, validate that fixation/permeabilization doesn't affect membrane epitopes

• For phospho-NDRG1 detection, include phosphatase inhibitors in all buffers and process samples rapidly

Analytical approaches:

• Quantify mean fluorescence intensity (MFI) rather than percent positive cells for more precise measurement

• For phosphorylation studies, calculate fold change in MFI relative to unstimulated controls

• Consider dimensionality reduction techniques (tSNE, UMAP) for complex datasets examining NDRG1 in heterogeneous populations

These strategies enable robust multiparameter analysis of NDRG1 expression and phosphorylation in relation to other cellular markers, providing deeper insights into its functional roles in different cell types and disease states.

How can researchers optimize imaging protocols when studying NDRG1 in tissues with high autofluorescence?

Tissue autofluorescence, particularly in the green spectrum where FITC emits, can significantly hinder specific signal detection. Optimization strategies include:

Autofluorescence reduction treatments:

• Sodium borohydride (0.1% in PBS, 30 minutes) reduces aldehyde-induced autofluorescence

• Sudan Black B (0.1-0.3% in 70% ethanol) quenches lipofuscin autofluorescence

• Photobleaching samples prior to antibody application can reduce endogenous fluorescence

Alternative fluorophore selection:

• Consider far-red or near-infrared conjugates (AF647, AF680, AF750) which emit in spectral regions with typically lower tissue autofluorescence

• These longer-wavelength fluorophores generally provide better signal-to-noise ratios in autofluorescent tissues

Advanced microscopy techniques:

• Spectral imaging combined with linear unmixing algorithms can separate specific signals from autofluorescence

• Confocal microscopy with narrow bandwidth detection focused on peak FITC emission

• Time-gated detection can exploit differences in fluorescence lifetime between specific signal and autofluorescence

Optimized blocking and staining protocols:

• Extended blocking (2-3 hours) with combinations of serum, BSA, and non-fat dry milk

• Increased antibody concentration may be necessary to overcome background

• Thorough washing steps with 0.1% Tween-20 to reduce non-specific binding

These approaches can significantly improve the detection of specific NDRG1 signals in challenging tissue samples, enabling more reliable analysis of its expression and localization in normal and pathological conditions.

How can FITC-conjugated NDRG1 antibodies contribute to understanding NDRG1's role in cancer?

NDRG1 functions as a tumor suppressor in many cell types and influences response to targeted therapies, making it an important subject in cancer research:

Tumor microenvironment studies:

• Multiparameter analysis of NDRG1 expression in different cellular components of tumors

• Correlation with markers of hypoxia, inflammation, and immune cell infiltration

• Understanding how tumor microenvironmental factors regulate NDRG1 expression and phosphorylation

Therapy response prediction:

• Flow cytometric quantification of NDRG1 in patient samples before treatment

• Correlation with response to EGFR-targeted therapies like cetuximab

• The Nature study suggests NDRG1 expression levels might serve as a predictive biomarker for cetuximab sensitivity in colorectal cancer

Metastasis and invasion mechanisms:

• NDRG1 has been associated with regulation of cancer cell invasion and metastasis

• Studies could examine how NDRG1 phosphorylation states correlate with metastatic potential

• Analysis of NDRG1's interaction with cytoskeletal proteins and cell adhesion molecules

Therapeutic targeting approaches:

• Screening compounds that modulate NDRG1 expression or phosphorylation

• Iron chelators can activate NDRG1 and might sensitize resistant tumors to therapy

• Investigating combination approaches targeting NDRG1 regulatory pathways

These research directions could advance understanding of NDRG1's complex roles in cancer biology and potentially lead to new diagnostic or therapeutic approaches.

What future applications might emerge for studying NDRG1 in vascular diseases?

The AHA Journals article highlights NDRG1's critical importance in endothelial inflammation and thrombotic responses, suggesting several promising research directions:

Atherosclerosis progression studies:

• Endothelial cell-specific NDRG1 knockout mice showed markedly attenuated atherosclerosis formation

• Analysis of NDRG1 expression and phosphorylation in different stages of atherosclerotic plaque development

• Investigation of NDRG1's role in endothelial-to-mesenchymal transition during vascular remodeling

Thrombosis and coagulation research:

• NDRG1 knockdown affected expression of procoagulant molecules (PAI-1, TF) and increased expression of TM and t-PA

• Studies could explore NDRG1's role in regulating the balance between pro- and anti-thrombotic factors

• Analysis of NDRG1 levels in patients with thrombotic disorders

Therapeutic modulation strategies:

• Development of endothelial-targeted NDRG1 modulators for vascular disease treatment

• Investigation of existing drugs that might affect NDRG1 expression or activity in endothelial cells

• Exploration of NDRG1's interaction with Nur77 as a potential intervention point

Mechanotransduction research:

• Since NDRG1 expression is regulated by fluid shear stress, studies could investigate its role in vascular responses to altered hemodynamics

• Analysis of NDRG1's contribution to flow-dependent vascular remodeling

• Correlation of NDRG1 expression with regions of disturbed flow in vascular disease models

These emerging research directions could significantly advance understanding of NDRG1's role in vascular biology and potentially identify new therapeutic targets for inflammatory vascular diseases.