NGFR Antibody

What is NGFR and why is it significant in research?

NGFR (p75NTR) is a 75 kDa transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily. It functions as a low-affinity receptor for multiple neurotrophins (NGF, BDNF, NT-3, NT-4/5) and plays critical roles in neuronal growth, migration, differentiation, and programmed cell death . Beyond the nervous system, NGFR serves as a marker for multiple cell types, including:

• Neural crest-derived stem cells

• Mesenchymal stem cells

• Myoepithelial cells in breast tissue

• Epithelial basal cells in respiratory tissues

• Melanoma subpopulations associated with therapy resistance

NGFR's biological significance extends to both developmental processes and pathological conditions, making antibodies against this receptor valuable tools for investigating diverse cellular mechanisms .

Which applications are most validated for NGFR antibodies?

NGFR antibodies have been validated across multiple experimental platforms, with application-specific considerations:

Researchers should note that detection sensitivity varies significantly between applications, with immunohistochemistry and flow cytometry generally showing the highest robustness across different antibody clones .

How should NGFR antibodies be validated for experimental use?

A robust validation strategy for NGFR antibodies should include multiple complementary approaches:

• Epitope verification: Confirm the specific binding region on NGFR (important as different domains have distinct functions)

• Cross-reactivity assessment: Test against related proteins and across species - many NGFR antibodies show species-specific reactivity patterns

• Positive control tissues:

  • Neural tissues (peripheral nerves, sensory ganglia)

  • Melanoma samples (particularly for human studies)

  • Breast myoepithelial cells

  • Neuroblastoma cell lines

• Negative controls: Test on tissues known to lack NGFR expression and use isotype controls in flow cytometry

• Protein array validation: Several commercial antibodies have been validated against >19,000 human proteins using protein microarrays to ensure specificity

• Knockdown/knockout validation: When possible, verify antibody specificity in NGFR-depleted systems to confirm signal loss

For research requiring absolute specificity, combining detection methods (e.g., immunoblotting followed by immunofluorescence) provides stronger validation than single-method approaches .

What are the key considerations for immunohistochemical detection of NGFR?

NGFR immunohistochemistry requires particular attention to protocol optimization:

• Fixation effects: Paraformaldehyde/formalin fixation preserves NGFR epitopes, but excessive fixation can mask binding sites

• Epitope retrieval: Most protocols recommend:

  • Heat-induced epitope retrieval at pH 6.0 for 10-20 minutes

  • Alternative protocol: pH 9.0 for 10-30 minutes for certain antibody clones

• Antibody concentration: Optimal range typically 1-3 μg/ml or dilutions of 1:100-1:200 for concentrated antibodies

• Incubation conditions: Room temperature incubation for 30 minutes usually sufficient; overnight incubation at 4°C may increase sensitivity with reduced background

• Signal amplification: DAB detection with HRP polymer provides excellent signal-to-noise ratio for most applications

• Clinical applications: NGFR antibodies are particularly valuable for diagnosing desmoplastic and neurotrophic malignant melanomas when used with S100 antibodies, as these tumors often lack conventional melanocytic markers

Researchers should always include positive control tissues with known NGFR expression patterns when establishing new IHC protocols .

What are the optimal protocols for flow cytometric analysis using NGFR antibodies?

For flow cytometry applications with NGFR antibodies, researchers should consider:

• Clone selection:

  • NGFR5 (originally C34C): Recognizes epitope within amino acids 1-160

  • ME20.4: Binds epitope in third/fourth CRD of extracellular domain

  • 192-IgG: Well-characterized for rat p75NTR

• Conjugate options:

  • APC conjugates: Provide excellent sensitivity in multi-parameter experiments

  • Alexa Fluor 488/647: Lower background and photobleaching for sorting applications

• Buffer formulation: Include sodium azide (0.1%) to prevent receptor internalization during processing

• Titration: Always determine optimal antibody concentration empirically; under- or over-staining affects separation of positive populations

• Controls:

  • Fluorescence-minus-one (FMO) controls particularly important for NGFR due to variable expression levels

  • Isotype controls to account for non-specific binding

• Applications in CAR-T cell research: NGFR-derived hinges in CAR constructs can be detected using anti-NGFR antibodies; the N3 and N4 NGFR-derived hinge regions show superior detection compared to N1 and N2 variants

For isolation purposes, optimized MACS protocols using MS columns and Select microbeads have demonstrated excellent CAR-T cell purity and yield when using N3 and N4-hinged constructs .

How do different NGFR antibody clones compare in specificity and application suitability?

Researchers should consider clone-specific characteristics when selecting NGFR antibodies:

Clone selection should be guided by the specific experimental context, with particular attention to species reactivity and the intended application .

How can NGFR antibodies facilitate the study of CAR-T cell technologies?

NGFR antibodies have become instrumental in CAR-T cell research through several applications:

• Detection and enrichment: NGFR-derived hinge regions incorporated into CAR constructs allow detection and high-grade enrichment of CAR-T cells using GMP-compatible immunomagnetic reagents

• Optimized hinge design: Two novel hinge domains (N3 and N4) derived from NGFR show superior properties:

  • N3 (120 aa): Contains third and fourth CRD plus S/T-rich stalk

  • N4 (162 aa): Additionally contains the second CRD

  • Both enable efficient selection on MACS MS columns with excellent CAR T cell purity and yield

• Efficacy comparisons: N3- and N4-hinged CAR T cells demonstrate comparable efficacy to CD8-hinged counterparts:

  • Against hematological blasts in vitro

  • In controlling acute monocytic leukemia in immunodeficient mouse xenograft models

• Selection optimization: When using NGFR-based selection:

  • MS columns with Select microbeads provide optimal results

  • Other column/microbead combinations (LS/LD with Standard/Select) result in higher losses of transduced T cells

• Expression detection: Using flow cytometry with ME20.4-PE antibody allows quantification of CAR expression levels on transduced cells

This technology represents an important advance in CAR-T cell manufacturing, potentially applicable across multiple clinical constructs .

What role does NGFR play in tumor biology and how can antibodies help investigate this?

NGFR has emerged as a significant player in tumor biology, particularly in therapy resistance:

• Cancer-specific expression:

  • Expressed in neural crest-derived tumors: melanomas, neuroblastomas, pheochromocytomas

  • Also detected in breast carcinoma and certain prostate cancers

• Therapy resistance mechanisms:

  • In melanoma, a pre-existing NGFR-high (NGFR^hi) subpopulation shows resistance to multiple therapies

  • These cells are refractory to cytotoxic T cells recognizing both differentiation and non-differentiation antigens

  • NGFR^hi cells also show resistance to BRAF+MEK inhibitors

• Immunomodulatory effects:

  • NGFR^hi tumor fractions associate with immune exclusion

  • NGFR-high cells induce BDNF (Brain-Derived Neurotrophic Factor), contributing to T cell resistance

  • A tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance in melanoma patients

• Intervention potential:

  • Pharmacologic NGFR inhibition can restore tumor sensitivity to T cell attack in vitro and in melanoma xenografts

  • This suggests NGFR as a potential therapeutic target to overcome resistance

NGFR antibodies enable researchers to identify and isolate these therapy-resistant subpopulations for mechanistic studies and targeted intervention development .

How should researchers interpret variable or conflicting results obtained with different NGFR antibodies?

Variability in NGFR antibody performance can arise from multiple factors:

• Epitope accessibility:

  • The extracellular domain of NGFR contains four cysteine-rich domains (CRDs) with different accessibility

  • Conformational changes upon ligand binding can mask certain epitopes

  • Protocols may need adjustment to expose specific binding regions

• Truncated forms:

  • Naturally occurring truncated NGFR containing only the extracellular domain exists in vivo

  • These forms are detected in urine, plasma, and amniotic fluid

  • Antibodies targeting different domains may differentially detect these variants

• Species-specific variations:

  • Sequence differences between species affect epitope conservation

  • Some antibodies (e.g., NGFR5) explicitly do not cross-react with mouse/rat NGFR

  • Always verify species reactivity for cross-species studies

• Application-specific performance:

  • An antibody performing well in IHC may not be optimal for Western blotting

  • For example, Thermo Fisher's MA5-13314 "does not react with rat tissue in Western blot applications" despite detecting rat NGFR in other applications

• Expression heterogeneity:

  • NGFR expression varies dramatically across different cell types and conditions

  • Negative results should be interpreted cautiously without positive controls

When encountering discrepancies, researchers should consider validating findings with multiple antibody clones targeting different epitopes and employing complementary detection methods .

What are the cutting-edge applications of NGFR antibodies in neuroscience research?

NGFR antibodies are enabling several advanced research directions in neuroscience:

• Neurodegeneration mechanisms:

  • Investigating NGFR's role in Alzheimer's Disease pathogenesis

  • Studying neuronal loss in the basal forebrain where NGFR is prominently expressed

  • Examining the balance between trophic and apoptotic signaling through NGFR in degenerative conditions

• Neural stem cell biology:

  • Isolation of neural crest stem cells based on NGFR expression

  • Lineage tracing of NGFR-positive progenitors during development and regeneration

  • Studying the differentiation potential of neuroepithelial-derived NGFR-positive cells into neurons, smooth muscle, and Schwann cells

• Neurotrophin signaling complexities:

  • Investigating the dual role of NGFR in promoting both survival and apoptosis

  • Examining NGFR signaling through multiple pathways (sphingomyelinase/ceramide, JNK, NF-κB)

  • Studying co-receptor interactions with Trks and their impact on signaling outcomes

• Regenerative medicine:

  • Identifying and characterizing NGFR-positive cells with regenerative potential

  • Studying NGFR's role in peripheral nerve regeneration

  • Developing strategies to modulate NGFR signaling for therapeutic benefit

These applications benefit from advances in NGFR antibody technology, including the development of highly specific monoclonal antibodies and fluorophore-conjugated variants for multiparameter analysis .

What methodological challenges exist in quantifying NGFR expression levels?

Accurate quantification of NGFR presents several methodological challenges:

• Heterogeneous expression:

  • NGFR often shows heterogeneous expression within tissues and cell populations

  • Single-cell techniques may be necessary to capture this heterogeneity effectively

• Standard curve development:

  • Recombinant NGFR standards exist but may not perfectly mimic native protein conformation

  • Quantitative assays should include internal calibrators representing different expression levels

• Western blot quantification limitations:

  • NGFR often shows multiple bands reflecting glycosylation variants and proteolytic processing

  • Normalization strategy selection critically affects quantification accuracy

• Flow cytometry considerations:

  • Receptor internalization during processing can affect surface detection

  • Antibody-binding capacity (ABC) beads recommended for standardizing mean fluorescence intensity values

• Absolute vs. relative quantification:

  • Relative comparisons between experimental conditions are more reliable than absolute quantification

  • When absolute quantities are required, digital PCR for mRNA quantification may complement protein detection methods

For comparative studies, consistent methodology is essential, including identical antibody clones, concentrations, and detection systems across all experimental groups .

How can researchers optimize NGFR antibody-based cell isolation protocols?

For optimal cell isolation using NGFR antibodies, researchers should consider:

• MACS optimization for NGFR-based selection:

  • Column type significantly affects yield: MS columns generally superior to LS or LD for NGFR-positive cells

  • Microbead conjugation matters: "Select" microbeads perform better than "Standard" for certain applications

  • Systematic testing of column/microbead combinations recommended for new applications

• Flow sorting considerations:

  • Conjugated antibodies reduce processing steps and improve viability

  • Alexa Fluor 488-conjugated antibodies (like 192-IgG) provide excellent signal-to-noise ratio

  • Titration of antibody concentration critical to distinguish positive from negative populations

• Buffer formulation:

  • Include protein (0.5-1% BSA) to reduce non-specific binding

  • For FACS, adding EDTA (2mM) prevents cell clumping

  • For MACS, buffer degassing improves column performance

• Post-isolation validation:

  • Always verify purity post-selection (typically by flow cytometry)

  • Check cell viability as isolation procedures can affect cell health

  • Perform functional assays to confirm isolated cells maintain expected biological properties

• CAR-T cell applications:

  • N3 and N4 NGFR-hinged CAR constructs show superior performance for immunomagnetic selection

  • Optimized protocol achieves >95% purity with excellent yield using Select microbeads on MS columns

These approaches have been successfully applied to diverse cell types, including neural crest stem cells, melanoma subpopulations, and engineered T cells .

What controls are essential when designing experiments with NGFR antibodies?

Robust experimental design with NGFR antibodies requires comprehensive controls:

• Positive tissue/cell controls:

  • Neural tissues (peripheral nerves, dorsal root ganglia)

  • Melanoma tissue sections (for human studies)

  • Neuroblastoma cell lines (e.g., MOLM-14)

  • Breast myoepithelial cells

• Negative controls:

  • Isotype-matched irrelevant antibodies (essential for flow cytometry)

  • Tissues known to lack NGFR expression

  • When possible, NGFR knockout/knockdown samples

• Technical controls:

  • For IHC: Omission of primary antibody

  • For flow cytometry: Fluorescence-minus-one (FMO) controls

  • For MACS: Pre-MACS, flow-through, and post-MACS samples to assess selection efficiency

• Epitope competition controls:

  • Pre-incubation with recombinant NGFR to block specific binding

  • Particularly valuable when testing new antibody clones or applications

• Validation across methods:

  • Orthogonal validation using multiple detection techniques

  • For example, confirming IHC results with Western blotting or flow cytometry

• Species cross-reactivity controls:

  • Testing on known positive samples from relevant species

  • Particularly important as many NGFR antibodies show species-specific patterns

Proper controls not only validate results but also help troubleshoot technical issues and interpret complex expression patterns .

What recent innovations have emerged in NGFR antibody development and applications?

Recent advancements in NGFR antibody technology include:

• Novel conjugates for multiparameter analysis:

  • Development of bright fluorophore conjugates (Alexa Fluor 647, APC)

  • Site-specific conjugation methods that preserve antibody function

  • Conjugation to non-fluorescent labels for mass cytometry (CyTOF) applications

• NGFR-derived hinge domains for cell engineering:

  • Creation of novel N3 and N4 NGFR-derived hinges for CAR-T cell constructs

  • These domains enable both detection and efficient selection of engineered cells

  • Demonstrated functional equivalence to clinically approved hinges (CD8-derived)

• Application in therapy resistance studies:

  • Identification of NGFR-high subpopulations in melanoma with multi-therapy resistance

  • Development of methods to isolate and characterize these cells

  • Demonstration that pharmacologic NGFR inhibition can reverse resistance phenotypes

• Advanced validation techniques:

  • Protein array validation against >19,000 human proteins

  • Z-score analysis to quantify binding specificity

  • Cross-correlation with genomic data to validate expression patterns

• Single-cell applications:

  • Integration with single-cell RNA-seq for correlation of protein and mRNA expression

  • Application in dissecting heterogeneity within NGFR-positive populations

  • Identification of novel NGFR-expressing subpopulations in complex tissues

These innovations are expanding the utility of NGFR antibodies beyond traditional applications into emerging fields like cellular therapy and precision medicine .

How should researchers approach NGFR antibody selection for multi-species studies?

Cross-species studies using NGFR antibodies require careful consideration:

• Species-specific epitope conservation:

  • Human NGFR shares 93% amino acid identity with non-human primates but only ~80% with rodents

  • Extracellular domain epitopes tend to be more conserved than intracellular regions

  • Many commercial antibodies have defined species reactivity patterns:

    • Some react only with human NGFR

    • Some cross-react with non-human primates, rabbit, cat, and ferret

    • Some explicitly do not cross-react with mouse and rat NGFR

• Validation strategy for each species:

  • Verify reactivity in each species independently

  • Use species-specific positive controls

  • Do not assume cross-reactivity based on sequence homology alone

• Application-specific considerations:

  • An antibody may cross-react in some applications but not others

  • For example, the MA5-13314 antibody shows reactivity with rat samples in flow cytometry but not in Western blot

• Alternative approaches when cross-reactivity is limited:

  • Use species-specific antibodies (e.g., 192-IgG for rat studies)

  • Consider epitope tagging strategies for engineered expression systems

  • Use mRNA detection methods as complementary approaches

• Documented cross-reactivity combinations:

  • NGFR5 clone: Human, non-human primate, rabbit, cat, ferret (does not react with mouse/rat)

  • Select antibodies from R&D Systems: Human, mouse, rat, pig

  • Rabbit polyclonal antibodies often show broader cross-reactivity