NDRG4 Antibody

What is the molecular characterization of NDRG4 protein targeted by commercial antibodies?

NDRG4 (NDRG family member 4) is a 339 amino acid protein with a calculated molecular weight of 37 kDa, though it is commonly observed at 37 kDa, 38 kDa, and 41 kDa in Western blot applications due to post-translational modifications and alternative splicing . Unlike other NDRG family members that show widespread expression, NDRG4 expression is primarily restricted to the heart and brain tissues . Multiple alternatively spliced isoforms of NDRG4 have been reported in the literature, which may explain the variation in observed molecular weights on Western blots . The gene is identified by NCBI Gene ID 65009 and UniProt ID Q9ULP0, with GenBank accession number BC011795 .

Which experimental applications are validated for NDRG4 antibodies?

NDRG4 antibodies have been validated for multiple experimental applications with specific recommended dilutions that vary by manufacturer and application type:

These applications have been validated in published literature, with Western blotting being the most commonly cited technique .

What is the species reactivity profile of commercially available NDRG4 antibodies?

Commercial NDRG4 antibodies demonstrate reactivity with human, mouse, and rat samples as confirmed through experimental validation . For Proteintech's 12184-1-AP antibody, positive Western blot detection has been specifically documented in human brain tissue and mouse kidney tissue . The antibody from Cell Signaling Technology (product #9039) also shows reactivity with human, mouse, and rat samples at the endogenous level . Some manufacturers may predict reactivity with additional species based on sequence homology, though this should be experimentally validated before use in critical applications .

What are the optimal storage and handling conditions for NDRG4 antibodies?

For maximum stability and activity retention, NDRG4 antibodies should be stored at -20°C where they remain stable for one year after shipment . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage according to manufacturer guidelines . Some formulations (particularly smaller sizes like 20μl) may contain 0.1% BSA as a stabilizer . When working with these antibodies, it's advisable to minimize freeze-thaw cycles and keep the antibody on ice during experiment preparation to preserve binding capacity and specificity .

How should antigen retrieval be optimized for NDRG4 immunohistochemistry?

Optimizing antigen retrieval is critical for NDRG4 immunohistochemistry due to the tissue-specific expression pattern of this protein. Based on validated protocols, two primary antigen retrieval methods are recommended:

• Primary recommendation: TE buffer pH 9.0, which has shown superior results in human medulloblastoma tissue, human colon tissue, and human gliomas tissue .

• Alternative method: Citrate buffer pH 6.0, which can be used if the TE buffer method produces suboptimal results .

The optimal retrieval method may be tissue-dependent. For example, when working with brain tissues where NDRG4 is highly expressed, the TE buffer method typically produces clearer staining with less background. For heart tissues, a careful titration between both methods is advisable. Researchers should perform a systematic comparison of both methods with their specific tissue samples before proceeding with large-scale experiments .

What are the recommended positive controls for validating NDRG4 antibody specificity?

Based on the tissue-specific expression pattern of NDRG4, the following samples serve as reliable positive controls for antibody validation:

When validating a new lot of NDRG4 antibody, comparison with these established positive controls provides confidence in antibody specificity . For negative controls, tissues known to lack NDRG4 expression can be used, though siRNA knockdown in cell lines that express NDRG4 would provide more definitive evidence of specificity . The staining pattern should match the expected subcellular localization of NDRG4 based on published literature .

How should dilution optimization be approached for Western blot analysis of NDRG4?

Optimizing antibody dilution for Western blot analysis of NDRG4 requires systematic titration within the recommended range (1:500-1:3000) . The process should follow these methodological steps:

• Begin with a middle-range dilution (e.g., 1:1000) using a known positive control (human brain tissue or mouse kidney tissue).

• Evaluate signal-to-noise ratio, examining both the clarity of bands at the expected molecular weights (37 kDa, 38 kDa, and 41 kDa) and the presence of non-specific background.

• If signal is weak but specific, decrease the dilution (e.g., to 1:500); if signal is strong but with high background, increase the dilution (e.g., to 1:2000).

• For tissues with lower NDRG4 expression, longer exposure times and lower dilutions may be necessary, while maintaining overnight primary antibody incubation at 4°C.

• Document the optimal dilution for each tissue type and experimental condition, as the optimal dilution may vary based on protein extraction method, sample type, and detection system .

How can NDRG4 antibodies be utilized to study its role in myogenesis and muscle differentiation?

NDRG4 plays a significant role in myogenesis, dramatically promoting the expression of myogenic differentiation factors (MyoD, MyoG) and myosin heavy chain (MyHC) genes while enhancing myotube formation . To investigate this function:

• Experimental design approach: Combine NDRG4 antibody-based protein detection with functional studies involving NDRG4 knockdown or overexpression in C2C12 myoblasts:

  • Transfect cells with NDRG4 siRNA or overexpression vectors

  • Induce differentiation (typically by serum withdrawal)

  • Harvest cells at defined timepoints (days 0, 2, 4 post-differentiation)

  • Use Western blotting with NDRG4 antibody (1:1000 dilution) alongside antibodies for MyoD, MyoG, and MyHC

  • Perform immunofluorescence for MyHC to quantify myotube formation

• Data analysis approach: Quantify:

  • Percentage of mononucleate cells

  • Percentage of cells with 2-5 nuclei

  • Percentage of cells with ≥6 nuclei

• Interpretation framework: NDRG4 knockdown typically increases mononucleate cells (54% vs. 31% in controls) while decreasing multinucleated myotubes. Conversely, NDRG4 overexpression enhances myotube formation and size .

This methodological approach allows for mechanistic studies of NDRG4's functional role in the differentiation process, particularly in relation to the Akt/CREB activation pathway implicated in myogenesis .

What methodological considerations are important when investigating NDRG4 hypermethylation as a cancer biomarker?

NDRG4 promoter hypermethylation represents a promising mechanistic biomarker for metastatic potential in several cancers, including breast cancer . When designing studies to investigate this epigenetic modification:

• Tissue sample selection: Include matched pairs of primary tumor and metastatic tissue from the same patients whenever possible. For breast cancer specifically, comparing lymph node-negative versus lymph node-positive cases provides valuable insights into metastatic potential .

• Methodological workflow:

  • Extract DNA from tumor tissues

  • Perform bisulfite conversion (critical for methylation analysis)

  • Use methylation-specific PCR or bisulfite sequencing to assess NDRG4 promoter methylation status

  • Complement with NDRG4 protein expression analysis via IHC (1:50-1:500 dilution)

  • Include functional assays examining cell adhesion to fibronectin and vitronectin

• Critical controls:

  • Adjacent normal tissue as methylation baseline

  • Commercially available methylated and unmethylated DNA standards

  • Cell lines with known NDRG4 methylation status

• Interpretation framework: Aberrant NDRG4 hypermethylation is associated with:

  • Downregulation of NDRG4 transcription and protein expression

  • Enhanced lymph node adhesion and cell mobility

  • Modulation of integrin signaling (particularly β1-integrins)

  • "Adhesive switch" phenotype (decreased adhesion to fibronectin, increased adhesion to vitronectin)

These methodological considerations ensure robust assessment of NDRG4 methylation status and its functional consequences in cancer progression .

How can NDRG4 antibodies be employed to investigate its role in neurological disorders?

NDRG4 expression is reduced in the brains of patients with Alzheimer's disease, suggesting a potential role in neurological function . To investigate this connection:

• Tissue-specific examination:

  • Use NDRG4 antibodies for IHC (1:800 dilution) on brain sections from control and Alzheimer's disease patients

  • Focus on regions most affected in Alzheimer's disease (hippocampus, entorhinal cortex)

  • Perform co-staining with markers for neurons, astrocytes, and microglia to determine cell type-specific expression

• Quantification approach:

  • Use digital image analysis to quantify NDRG4 expression levels

  • Correlate with disease severity markers and cognitive assessment scores

  • Compare with other NDRG family members to assess specificity

• Mechanistic investigations:

  • Complement protein expression studies with functional assays in neuronal cell models

  • Evaluate NDRG4's interaction with proteins involved in neurodegeneration

  • Investigate potential neuroprotective functions through knockdown/overexpression studies

This methodological approach can help elucidate whether NDRG4 reduction is a cause or consequence of neurodegeneration, and whether it represents a potential therapeutic target for Alzheimer's disease .

How should researchers address multiple bands observed in Western blots for NDRG4?

When Western blotting for NDRG4, researchers often observe multiple bands at approximately 37 kDa, 38 kDa, and 41 kDa . This is not necessarily an indication of antibody non-specificity, but rather reflects the biological complexity of NDRG4. To address this technical challenge:

• Verification approach:

  • Compare observed band pattern with expected molecular weights (37-45 kDa range)

  • Verify using positive controls (human brain tissue, mouse kidney tissue)

  • Run lysates from tissues known to not express NDRG4 as negative controls

  • Perform validation with NDRG4 knockdown/overexpression if available

• Interpretation framework:

  • Multiple bands likely represent different isoforms of NDRG4 (multiple alternatively spliced isoforms have been reported)

  • Post-translational modifications may also contribute to size variations

  • The pattern of bands may vary by tissue type, reflecting tissue-specific isoform expression

• Documentation practice:

  • Report all observed bands within the expected molecular weight range

  • Include positive controls on all blots for reference

  • Document exposure time and detection method used

By following this systematic approach, researchers can confidently distinguish genuine NDRG4 signal from non-specific antibody binding .

What strategies can address weak or inconsistent NDRG4 staining in immunohistochemistry?

When experiencing weak or inconsistent NDRG4 staining in immunohistochemistry, consider this methodological approach:

• Antigen retrieval optimization:

  • Compare TE buffer pH 9.0 versus citrate buffer pH 6.0

  • Extend retrieval time (15-20 minutes) for formalin-fixed tissues

  • Consider testing pressure cooker versus microwave-based retrieval methods

• Antibody incubation parameters:

  • Decrease dilution (e.g., from 1:500 to 1:50) for tissues with lower expression

  • Extend primary antibody incubation (overnight at 4°C versus 1 hour at room temperature)

  • Test different detection systems (polymer-based versus avidin-biotin)

• Sample-specific considerations:

  • Fixation time can significantly impact epitope accessibility

  • Tissue-specific optimization may be necessary due to NDRG4's restricted expression pattern

  • Positive control sections (human medulloblastoma, colon, or gliomas tissue) should be included in each staining run

• Signal amplification strategies:

  • Consider tyramide signal amplification for very low expression levels

  • Evaluate alternative NDRG4 antibodies if persistent issues occur

  • Document optimized conditions for specific tissue types

These strategies address the most common technical issues in NDRG4 immunohistochemistry while maintaining staining specificity .

How can researchers validate knockdown efficiency when using NDRG4 siRNA in functional studies?

When performing NDRG4 knockdown studies, rigorous validation of knockdown efficiency is critical for reliable interpretation of functional outcomes. Researchers should employ the following methodological approach:

• Multi-level validation strategy:

  • Western blot using validated NDRG4 antibody (1:1000 dilution) to quantify protein reduction

  • qRT-PCR to measure mRNA expression changes

  • Immunofluorescence to visualize cellular expression patterns

• Quantification approach:

  • Normalize NDRG4 protein levels to appropriate loading controls (β-actin, GAPDH)

  • Calculate percentage knockdown relative to control (non-targeting siRNA)

  • Document time course of knockdown (typically day 0, 2, and 4 post-transfection for myogenic differentiation studies)

• Experimental design considerations:

  • Include multiple siRNA sequences targeting different regions of NDRG4

  • Test concentration-dependent effects (typically 10-50 nM range)

  • Assess potential off-target effects by examining other NDRG family members

• Functional correlation:

  • For myogenesis studies, correlate knockdown efficiency with changes in MyoD, MyoG, and MyHC expression

  • Quantify morphological changes (e.g., myotube formation) in relation to knockdown level

  • Document temporal relationship between NDRG4 reduction and functional consequences

How do NDRG4 expression patterns differ between cancer types, and what methodological approaches best capture these differences?

NDRG4 exhibits complex, cancer type-specific expression patterns that require tailored methodological approaches for accurate analysis:

• Cancer-specific expression profiles:

  • Glioblastoma: Elevated NDRG4 expression contributes to cell cycle progression and survival

  • Colorectal cancer: NDRG4 often inactivated by promoter methylation, suggesting tumor suppressor function

  • Breast cancer: NDRG4 epigenetic silencing associated with metastatic potential

• Comprehensive analytical approach:

  • Multi-tissue microarrays: Use standardized IHC protocol (1:50-1:500 dilution) across cancer types

  • Scoring system: Implement semi-quantitative scoring (0-3+) with both intensity and percentage positive cells

  • Molecular correlation: Pair protein expression data with methylation status and mRNA levels

  • Subcellular localization: Document cytoplasmic versus nuclear staining patterns

• Application-specific methodology:

  • For methylation studies: Bisulfite sequencing of the NDRG4 promoter region

  • For functional studies: Cell type-specific knockdown/overexpression with appropriate cancer cell models

  • For prognostic value assessment: Correlate expression with patient outcome data

• Interpretation framework:

  • Context-dependent function (oncogenic versus tumor suppressive) based on cancer type

  • Integration of protein expression with epigenetic regulation mechanisms

  • Correlation with clinical parameters (stage, grade, metastatic status)

This methodological approach acknowledges the tissue-specific and context-dependent roles of NDRG4 in different cancer types .

What methodological considerations are important when investigating NDRG4's impact on integrin signaling and cell adhesion?

NDRG4 modulates integrin signaling by influencing β1-integrin clustering and cellular adhesion properties, a mechanism particularly relevant in metastatic potential . To properly investigate this function:

• Adhesion assay methodology:

  • Compare adhesion to different extracellular matrix components (fibronectin versus vitronectin)

  • Quantify adhesion at multiple timepoints (15, 30, 60 minutes) following cell seeding

  • Include controls with function-blocking antibodies against specific integrin subunits

• Integrin clustering visualization:

  • Perform immunofluorescence using antibodies against β1-integrins

  • Document formation of "large punctate clusters" at the leading edge of cells

  • Quantify cluster size, number, and distribution using image analysis software

• Molecular mechanism investigation:

  • Assess activation status of downstream integrin signaling components

  • Examine co-localization of NDRG4 with integrin complexes

  • Evaluate effects of NDRG4 manipulation on integrin expression versus clustering

• Functional correlation with metastatic potential:

  • Pair adhesion assays with migration and invasion assays

  • Focus particularly on adhesion to vitronectin (important component of human lymph nodes)

  • Document the "adhesive switch" phenotype (decreased fibronectin adhesion, increased vitronectin adhesion)

This methodological approach provides mechanistic insights into how NDRG4 influences cell-matrix interactions and potentially contributes to metastatic behavior in cancer cells .

What methodological approaches can be used to investigate potential interactions between NDRG4 and other NDRG family members?

While NDRG4 exhibits tissue-specific expression compared to other more widely expressed NDRG family members, investigating potential functional interactions requires specialized methodological approaches:

• Co-expression analysis methodology:

  • Use multi-label immunofluorescence with validated antibodies against different NDRG family members

  • Focus on tissues where multiple family members are expressed (brain regions)

  • Quantify co-localization coefficients to determine spatial relationships

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation using NDRG4 antibody (1:100 dilution)

  • Confirm interactions with reciprocal immunoprecipitation

  • Consider proximity ligation assays for in situ detection of protein interactions

• Functional redundancy assessment:

  • Design combinatorial knockdown experiments (NDRG4 + other family members)

  • Compare phenotypic effects of single versus combined manipulation

  • Evaluate compensatory expression changes among family members

• Tissue-specific considerations:

  • Focus on heart and brain tissues where NDRG4 is primarily expressed

  • Investigate whether expression patterns change under pathological conditions

  • Consider developmental timepoints to assess temporal regulation

This methodological framework enables researchers to determine whether NDRG4 functions independently or in concert with other family members, providing insights into potential functional redundancy or complementarity.

How can researchers design experiments to elucidate the role of NDRG4 in therapeutic response?

Given NDRG4's involvement in cancer progression and cell survival, investigating its role in therapeutic response requires a systematic experimental approach:

• Cell model selection strategy:

  • Choose cell lines with defined NDRG4 expression levels (high in glioblastoma, variable in other cancers)

  • Generate stable NDRG4 knockdown and overexpression models

  • Include patient-derived cell lines to enhance clinical relevance

• Treatment-response methodology:

  • Test multiple therapeutic agents (conventional chemotherapy, targeted therapies)

  • Perform dose-response curves to determine IC50 values

  • Assess acute versus long-term responses (resistance development)

• Molecular response analysis:

  • Monitor NDRG4 expression changes following treatment using Western blot (1:1000 dilution)

  • Investigate downstream pathway activation (integrin signaling, survival pathways)

  • Correlate NDRG4 levels with markers of apoptosis and cell cycle arrest

• Translational research approach:

  • Analyze publicly available datasets for correlations between NDRG4 expression and treatment outcomes

  • Consider testing NDRG4 as a predictive biomarker in retrospective patient cohorts

  • Investigate combination approaches targeting NDRG4-related pathways

This experimental framework allows researchers to determine whether NDRG4 impacts therapeutic efficacy and potentially identify strategies to overcome NDRG4-mediated treatment resistance in cancer settings .

How should researchers interpret discrepancies between NDRG4 mRNA and protein expression levels in experimental studies?

When faced with discrepancies between NDRG4 mRNA and protein levels, researchers should consider this systematic analytical approach:

• Verification methodology:

  • Confirm antibody specificity through positive and negative controls

  • Validate primer specificity for distinguishing between NDRG4 isoforms

  • Consider using multiple antibodies targeting different epitopes

• Regulatory mechanism investigation:

  • Assess post-transcriptional regulation (microRNAs targeting NDRG4)

  • Examine protein stability and half-life through cycloheximide chase experiments

  • Investigate epigenetic regulation (DNA methylation, histone modifications)

• Isoform-specific analysis:

  • Design primers to detect specific NDRG4 splice variants

  • Compare protein detection pattern (multiple bands) with predicted isoform sizes

  • Consider tissue-specific expression patterns of different isoforms

• Integrated interpretation framework:

  • In cancer studies, consider promoter methylation as a cause of transcriptional silencing without affecting protein stability

  • In differentiation studies, examine temporal relationships between mRNA and protein changes

  • Document tissue-specific post-transcriptional regulatory mechanisms

This analytical approach acknowledges that discrepancies between mRNA and protein levels may reflect biologically relevant regulatory mechanisms rather than technical artifacts .

What statistical approaches are most appropriate for analyzing NDRG4 expression data in clinical samples?

When analyzing NDRG4 expression in clinical samples, appropriate statistical methodology is critical for robust interpretation:

• Data normalization strategy:

  • For Western blot: Normalize to loading controls and reference samples across blots

  • For IHC: Use standardized scoring systems (H-score, Allred score) that account for both intensity and percentage of positive cells

  • For qRT-PCR: Select stable reference genes validated for the specific tissue type

• Comparative analysis methodology:

  • For two-group comparisons (e.g., normal vs. tumor): Student's t-test or Mann-Whitney test depending on data distribution

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For paired samples (e.g., primary tumor vs. metastasis): Paired t-test or Wilcoxon signed-rank test

• Correlation analysis:

  • With continuous variables: Pearson's or Spearman's correlation coefficient

  • With categorical variables: Chi-square test or Fisher's exact test

  • With survival data: Kaplan-Meier analysis with log-rank test and Cox proportional hazards modeling

• Advanced analytical approaches:

  • Consider multivariate models adjusting for relevant clinicopathological factors

  • Implement receiver operating characteristic (ROC) analysis for biomarker performance assessment

  • For methylation studies, use appropriate methods for analyzing proportional data

These statistical approaches ensure rigorous analysis of NDRG4 expression data in clinical contexts, particularly when evaluating its potential as a biomarker .

What are the most promising future research directions for NDRG4 antibody applications in neurodegenerative disease research?

Based on current knowledge of NDRG4's reduced expression in Alzheimer's disease and its primarily brain-specific expression pattern , several promising research directions emerge:

• Biomarker development methodology:

  • Investigate NDRG4 levels in cerebrospinal fluid using sensitive immunoassays

  • Correlate NDRG4 expression patterns with neuroimaging findings

  • Examine potential for early detection before symptom onset

• Mechanistic research approach:

  • Explore NDRG4's role in neuronal survival and function

  • Investigate interactions with key proteins implicated in neurodegeneration

  • Develop conditional knockout models to assess brain-specific functions

• Therapeutic targeting strategy:

  • Explore methods to restore NDRG4 expression in affected brain regions

  • Investigate downstream pathways as potential therapeutic targets

  • Develop screening platforms for compounds that modulate NDRG4 function

• Translational research directions:

  • Expand studies to other neurodegenerative conditions beyond Alzheimer's

  • Investigate NDRG4 in models of neuronal injury and repair

  • Explore potential genetic associations with disease risk or progression

These research directions leverage NDRG4 antibodies as critical tools for advancing our understanding of NDRG4's role in neurological health and disease .

How might emerging technologies enhance the utility and applications of NDRG4 antibodies in research?

Emerging technologies offer significant opportunities to expand NDRG4 antibody applications and enhance their research utility:

• Single-cell analysis approaches:

  • Adapt NDRG4 antibodies for mass cytometry (CyTOF) applications

  • Implement imaging mass cytometry for spatial expression analysis in tissues

  • Develop NDRG4 antibodies compatible with single-cell Western blotting technologies

• High-throughput screening methodology:

  • Optimize NDRG4 antibodies for automated immunofluorescence platforms

  • Develop cell-based assays for compound screening affecting NDRG4 expression

  • Implement NDRG4 detection in organoid models for 3D expression analysis

• Advanced imaging techniques:

  • Adapt NDRG4 antibodies for super-resolution microscopy applications

  • Develop live-cell imaging approaches using non-disruptive labeling strategies

  • Implement multiplexed imaging to simultaneously detect NDRG4 and interacting partners

• In vivo applications:

  • Develop NDRG4 antibody fragments for improved tissue penetration

  • Explore antibody-based imaging probes for non-invasive detection

  • Consider therapeutic applications targeting NDRG4-expressing tumors