NTRK1 Antibody, HRP conjugated

What is NTRK1 and why is it an important research target?

NTRK1 (Neurotrophic Tyrosine Kinase Receptor Type 1), also known as TrkA, is a membrane-bound receptor encoded by the NTRK1 gene. It functions primarily in the nervous system and is critically involved in the regulation of cell proliferation, differentiation, and survival . NTRK1 activates its intrinsic tyrosine kinase activity by binding to neurotrophic factors, particularly nerve growth factor (NGF), thereby triggering intracellular signaling cascades including MAPK and PI3K/AKT pathways . These pathways are crucial for both normal cellular development and pathological processes such as tumorigenesis. Research on NTRK1 is particularly important due to its role in cancer biology, where NTRK1 gene fusions can drive tumor growth and metastasis, making it a significant therapeutic target in oncology research .

What are the key applications for NTRK1 antibodies conjugated with HRP?

NTRK1 antibodies conjugated with HRP are valuable tools in multiple laboratory techniques including:

• Western Blotting (WB): For protein detection and quantification

• Enzyme-Linked Immunosorbent Assay (ELISA): For sensitive protein detection in solution

• Immunohistochemistry with paraffin-embedded sections (IHC-P): For analyzing protein expression in preserved tissue samples

• Immunohistochemistry with frozen sections (IHC-F): For analyzing protein expression in frozen tissue specimens

The HRP conjugation eliminates the need for secondary antibody incubation, simplifying experimental workflows and potentially reducing background signals in these applications.

How can I verify the specificity of an NTRK1-HRP conjugated antibody?

Verifying antibody specificity is essential for reliable research outcomes. For NTRK1-HRP conjugated antibodies, consider these methodological approaches:

• Positive and negative control tissues/cell lines: Use samples known to express or lack NTRK1 (brain tissue typically shows high expression).

• Molecular weight verification: In Western blots, NTRK1 should appear at approximately 87.5 kDa, though multiple isoforms may be detected (88, 87, 84, 78 kDa) . Note that post-translational modifications can result in higher observed molecular weights (up to 145 kDa) .

• Knockout/knockdown validation: Compare signal in wild-type samples versus those where NTRK1 has been genetically depleted.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (for example, synthetic peptide from amino acids 101-200 of human TrkA) to confirm binding specificity.

• Cross-reactivity assessment: If working with non-human samples, confirm species reactivity, as some antibodies show proven reactivity with human and rat samples, with predicted reactivity to mouse, cow, sheep, pig, and horse models .

How should I optimize Western blotting protocols for NTRK1-HRP antibodies?

Optimization of Western blotting protocols for NTRK1-HRP conjugated antibodies requires careful attention to several parameters:

• Sample preparation:

  • Use appropriate lysis buffers containing phosphatase inhibitors to preserve phosphorylation states

  • Include protease inhibitors to prevent degradation

  • Maintain samples at 4°C during processing

• Loading and separation:

  • Load 20-40 μg of total protein per lane

  • Use 8-10% SDS-PAGE gels for optimal separation of NTRK1 (87.5-145 kDa)

  • Include molecular weight markers spanning 50-200 kDa range

• Transfer conditions:

  • Wet transfer is recommended for large proteins

  • Use PVDF membranes for better protein retention

  • Transfer at 30V overnight at 4°C for complete transfer of high molecular weight proteins

• Antibody dilution and incubation:

  • Start with 1:1000 dilution in 5% BSA or non-fat milk in TBST

  • Incubate overnight at 4°C with gentle agitation

  • Include positive controls such as brain tissue lysates from human or rat

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrate compatible with HRP

  • Begin with short exposure times (30 seconds) and increase as needed

  • Consider using signal enhancers if the target is expressed at low levels

What are the critical considerations for immunohistochemistry using NTRK1-HRP antibodies?

When performing immunohistochemistry with NTRK1-HRP conjugated antibodies, researchers should consider these critical factors:

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes) based on tissue type and fixation conditions

• Blocking parameters:

  • Block endogenous peroxidase activity with 0.3-3% H₂O₂ for 10-15 minutes

  • Block endogenous biotin if using avidin-biotin detection systems

  • Use species-appropriate serum or protein blocker (5-10% normal serum)

• Antibody titration:

  • Perform a dilution series to determine optimal concentration

  • Start with manufacturer's recommended dilution (often 1:100 to 1:500)

  • Include positive control tissues (neural tissues) in optimization

• Signal development:

  • Since the antibody is HRP-conjugated, use DAB or other HRP substrates directly

  • Monitor signal development microscopically to prevent overdevelopment

  • Stop the reaction with buffer wash when optimal signal-to-noise ratio is achieved

• Counterstaining considerations:

  • Use light hematoxylin counterstaining to maintain visibility of DAB signal

  • Consider nuclear versus cytoplasmic localization of NTRK1 when evaluating results

How can NTRK1-HRP antibodies be validated for detecting NTRK1 fusion proteins in cancer research?

NTRK1 gene fusions are significant oncogenic drivers in various tumors . Validating NTRK1-HRP antibodies for detecting these fusion proteins requires specialized approaches:

• Epitope accessibility analysis:

  • Determine whether the antibody epitope (e.g., amino acids 101-200) is retained in common fusion proteins

  • Select antibodies targeting domains typically preserved in fusion proteins

• Positive control selection:

  • Use cell lines known to harbor specific NTRK1 fusions (e.g., KM12 cells with TPM3-NTRK1 fusion)

  • Include patient-derived xenograft models harboring validated NTRK1 fusions

• Comparative methodologies:

  • Confirm findings with orthogonal detection methods such as FISH or RT-PCR

  • Compare results with pan-TRK antibodies that detect multiple NTRK family members

• Molecular weight verification:

  • NTRK1 fusion proteins may exhibit different molecular weights than wild-type NTRK1

  • Document apparent molecular weights and compare with theoretical predictions based on fusion partner size

• Functional validation:

  • Correlate antibody detection with downstream signaling activation (p-ERK, p-AKT)

  • Confirm signal reduction following treatment with NTRK inhibitors

How should I design experiments to investigate NTRK1 signaling using HRP-conjugated antibodies?

Designing robust experiments to investigate NTRK1 signaling requires comprehensive planning:

• Stimulation protocols:

  • Use NGF (50-100 ng/ml) to activate NTRK1 signaling

  • Perform time-course experiments (5 minutes to 24 hours) to capture both immediate and delayed responses

  • Include controls treated with vehicle or heat-inactivated NGF

• Inhibition studies:

  • Incorporate selective NTRK1 inhibitors as controls

  • Consider parallel knockdown experiments using siNTRK1 to confirm antibody specificity and pathway effects

  • Remember that NTRK1 inhibition decreases YAP-driven transcription and affects cancer cell proliferation and migration

• Downstream pathway analysis:

  • Monitor activation of both MAPK and PI3K/AKT pathways

  • Examine effects on the Hippo pathway components (LATS1, YAP)

  • Evaluate expression of YAP target genes (CTGF, CYR61, ANKRD1) following NTRK1 activation/inhibition

• Experimental readouts:

  • Cell proliferation assays

  • Migration/wound healing assays

  • Colony formation assays

  • Immunofluorescence for YAP nuclear localization

• Data analysis approach:

  • Quantify signal intensity across multiple experiments

  • Normalize to appropriate loading controls

  • Perform statistical analysis using ANOVA with post-hoc tests for time-course experiments

What are the potential pitfalls in interpreting results from NTRK1-HRP antibody experiments?

Interpreting results from experiments utilizing NTRK1-HRP conjugated antibodies requires awareness of several potential pitfalls:

• Molecular weight variability:

  • NTRK1 can appear at different molecular weights (predicted: 88, 87, 84, 78 kDa; observed: up to 145 kDa)

  • Post-translational modifications including glycosylation and phosphorylation affect mobility

  • Cleavage products might be detected depending on sample preparation methods

• Cross-reactivity considerations:

  • Some antibodies may cross-react with other TRK family members (NTRK2/TrkB, NTRK3/TrkC)

  • Verify specificity using appropriate positive and negative controls

  • Confirm findings with alternative antibody clones targeting different epitopes

• Signal interpretation challenges:

  • HRP enzyme activity can be affected by sample buffer components

  • Signal saturation can mask quantitative differences

  • Auto-oxidation of substrates can create false-positive signals

• Context-dependent expression:

  • NTRK1 expression levels vary dramatically between tissue types

  • Expression and activation patterns may differ between cancer and normal cells

  • Alternative splicing generates multiple isoforms with potentially different antibody reactivity

• Pathway crosstalk effects:

  • NTRK1 signaling interacts with multiple pathways including Hippo signaling

  • Changes in NTRK1 detection may reflect alterations in associated proteins rather than NTRK1 itself

  • Consider analysis of entire signaling networks rather than isolated proteins

How can I reconcile contradictory results when using NTRK1-HRP antibodies across different experimental systems?

When faced with contradictory results across experimental systems, consider these methodological approaches:

• Antibody validation strategy:

  • Re-validate antibody in each experimental system

  • Use multiple antibodies targeting different NTRK1 epitopes

  • Consider non-antibody-based detection methods as complementary approaches

• System-specific variables:

  • Cell type-specific expression of co-receptors and adaptor proteins

  • Differences in post-translational modification machinery

  • Variations in endogenous ligand production

• Technical reconciliation approaches:

  • Standardize sample preparation protocols across systems

  • Use recombinant standards for quantitative comparisons

  • Implement internal normalization controls specific to each system

• Molecular context analysis:

  • Evaluate NTRK1 detection in relation to activation state

  • Consider membrane localization versus cytoplasmic or nuclear pools

  • Examine expression of potential binding partners that may mask epitopes

• Integrated data assessment:

  • Triangulate findings using orthogonal techniques (e.g., transcriptomics, proteomics)

  • Develop computational models that account for system-specific variables

  • Consider biological relevance of observed differences rather than forcing concordance

How can NTRK1-HRP antibodies be employed in studying the role of NTRK1 in the Hippo signaling pathway?

Recent research has revealed a previously unrecognized relationship between NTRK1 and the Hippo signaling pathway . NTRK1-HRP conjugated antibodies can be valuable tools in exploring this relationship:

• Co-immunoprecipitation studies:

  • Use NTRK1 antibodies to pull down protein complexes

  • Analyze interactions with Hippo pathway components (LATS1/2, MST1/2, YAP)

  • Examine how these interactions change with NGF stimulation or NTRK1 inhibition

• Phosphorylation state analysis:

  • Monitor changes in p-YAP and p-LATS1 levels following NTRK1 activation/inhibition

  • NGF treatment decreases p-YAP and p-LATS1 levels in a time-dependent manner

  • NTRK1 inhibition increases LATS1 and YAP phosphorylation

• Subcellular localization studies:

  • Track YAP nuclear translocation following NGF stimulation

  • Examine NTRK1 and YAP co-localization patterns

  • Investigate cytoplasmic sequestration of YAP following NTRK1 inhibition

• Gene expression analysis:

  • Measure YAP target gene expression (CTGF, CYR61, ANKRD1) following NTRK1 modulation

  • Implement Chromatin Immunoprecipitation (ChIP) to assess YAP binding to target promoters

  • Correlate gene expression changes with phenotypic outcomes in cell proliferation and migration assays

• Functional rescue experiments:

  • Test whether YAP overexpression can rescue phenotypes caused by NTRK1 inhibition

  • Determine if constitutively active LATS1 mimics NTRK1 inhibition effects

  • Evaluate if constitutively active YAP (YAP-S127A) can overcome NTRK1 knockdown effects

What methodological considerations are important when using NTRK1-HRP antibodies in multi-parameter flow cytometry?

While HRP-conjugated antibodies are not typically used for flow cytometry due to the need for substrate addition, researchers sometimes adapt protocols or use comparable fluorophore-conjugated versions. Important considerations include:

• Panel design strategy:

  • Select fluorophores with minimal spectral overlap

  • Include markers for relevant cell populations (e.g., neural crest-derived cells)

  • Incorporate phospho-specific antibodies for downstream signaling components

• Sample preparation optimization:

  • Use gentle cell dissociation methods to preserve surface NTRK1

  • Implement fixation and permeabilization for detecting total (intracellular + surface) NTRK1

  • Consider specialized fixatives that preserve phospho-epitopes

• Compensation and controls:

  • Include single-stained controls for each fluorophore

  • Use isotype controls matched to NTRK1 antibody (rabbit IgG-HRP or equivalent)

  • Implement fluorescence-minus-one (FMO) controls to set gating boundaries

• Data analysis approach:

  • Use dimensionality reduction techniques (tSNE, UMAP) for complex datasets

  • Consider density-based clustering algorithms to identify cell populations

  • Correlate NTRK1 expression with other parameters using bivariate plots

• Validation strategies:

  • Confirm flow cytometry findings with microscopy or Western blotting

  • Use cell sorting followed by functional assays to validate populations

  • Compare results across multiple antibody clones or detection systems

How can NTRK1-HRP antibodies contribute to understanding NTRK1 fusion proteins in cancer research?

NTRK1 fusion genes are important oncogenic drivers in various cancers . HRP-conjugated NTRK1 antibodies can provide valuable insights in this research area:

• Fusion protein detection strategy:

  • Select antibodies targeting domains retained in fusion proteins (typically the kinase domain)

  • Use paired antibodies targeting both NTRK1 and common fusion partners

  • Implement multiplex staining approaches to simultaneously detect fusion partners

• Expression pattern characterization:

  • Analyze tissue microarrays spanning multiple tumor types

  • Compare expression patterns between fusion-positive and fusion-negative tumors

  • Correlate expression with clinical parameters and outcomes

• Functional consequence assessment:

  • Examine downstream pathway activation (MAPK, PI3K/AKT, Hippo)

  • Analyze cellular phenotypes (proliferation, migration, survival)

  • Evaluate response to targeted NTRK inhibitors

• Resistance mechanism investigation:

  • Monitor changes in NTRK1 fusion protein expression following treatment

  • Identify compensatory signaling pathways activated upon NTRK inhibition

  • Detect secondary mutations in the kinase domain that confer drug resistance

• Biomarker development approach:

  • Establish standardized protocols for fusion protein detection

  • Determine sensitivity and specificity of antibody-based detection compared to molecular methods

  • Develop algorithms integrating multiple biomarkers for patient stratification

What are the most common technical issues with NTRK1-HRP antibodies and how can they be resolved?

Researchers often encounter several technical challenges when working with NTRK1-HRP conjugated antibodies:

• High background signal:

  • Increase blocking time and concentration (5-10% normal serum or BSA)

  • Reduce antibody concentration (test dilutions from 1:500 to 1:5000)

  • Include additional washing steps with increased stringency

  • Pre-absorb antibody with non-specific proteins before use

  • Ensure endogenous peroxidase activity is effectively quenched

• Weak or absent signal:

  • Optimize antigen retrieval methods for IHC applications

  • Increase antibody concentration or incubation time

  • Verify sample preparation preserves NTRK1 epitopes

  • Confirm NTRK1 expression in your experimental system

  • Try alternative lysis buffers for Western blotting applications

• Multiple bands in Western blotting:

  • Determine which bands represent specific signal using positive/negative controls

  • Consider that different isoforms may be present (predicted sizes: 88, 87, 84, 78 kDa)

  • Be aware that post-translational modifications can increase apparent molecular weight (up to 145 kDa)

  • Use phosphatase treatment to determine if bands represent phosphorylated forms

  • Optimize sample preparation to minimize protein degradation

• Inconsistent results between experiments:

  • Standardize all protocol parameters (times, temperatures, reagent concentrations)

  • Prepare fresh working solutions for each experiment

  • Consider lot-to-lot variations in antibodies

  • Implement positive controls in each experiment

  • Document all experimental conditions meticulously

• Cross-reactivity issues:

  • Validate antibody specificity in your experimental system

  • Use knockout/knockdown controls when possible

  • Consider competitive blocking with immunizing peptide

  • Compare results with alternative antibody clones

How can I optimize NTRK1-HRP antibody protocols for challenging experimental conditions?

Optimizing protocols for challenging conditions requires methodical troubleshooting:

• Fixed tissue samples with potential epitope masking:

  • Implement extended antigen retrieval (15-30 minutes)

  • Test multiple retrieval buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Consider proteolytic digestion as an alternative to heat-induced retrieval

  • Use signal amplification systems compatible with HRP

  • Extend primary antibody incubation time (overnight at 4°C)

• Low abundance targets:

  • Enrich for NTRK1-expressing cells before analysis (e.g., cell sorting)

  • Implement tyramide signal amplification for IHC applications

  • Use concentrated protein samples for Western blotting

  • Consider immunoprecipitation before Western blotting

  • Increase exposure time for Western blot imaging

• High lipid content samples:

  • Optimize tissue processing to remove excess lipids

  • Include detergents in antibody diluent (0.1-0.3% Triton X-100)

  • Use dewaxing solutions for paraffin sections

  • Implement additional blocking steps with non-fat milk

  • Consider specialized fixatives for lipid-rich tissues

• Samples with high endogenous peroxidase activity:

  • Extend peroxidase quenching steps (3% H₂O₂, 15-30 minutes)

  • Consider using alternative detection systems

  • Implement dual quenching with H₂O₂ and sodium azide

  • Use specialized blocking reagents for endogenous enzyme activity

  • Test fluorescent secondary antibodies as an alternative

• Degraded or archival samples:

  • Reduce antigen retrieval time to prevent further epitope degradation

  • Use antibody cocktails targeting multiple NTRK1 epitopes

  • Implement specialized retrieval protocols for archival materials

  • Consider alternative fixation methods for prospective samples

  • Use robust housekeeping proteins as controls for sample quality

What are the best practices for quantitative analysis of NTRK1 expression using HRP-conjugated antibodies?

Quantitative analysis of NTRK1 expression requires rigorous methodological approaches:

• Western blot quantification:

  • Use gradient gels for better separation of NTRK1 isoforms

  • Include recombinant NTRK1 standards at known concentrations

  • Implement loading controls appropriate for your experimental conditions

  • Use software with linear dynamic range detection capabilities

  • Perform multiple exposures to ensure measurements within linear range

  • Calculate relative density compared to housekeeping proteins

• IHC quantification:

  • Use digital image analysis software with validated algorithms

  • Implement H-score methodology (intensity × percentage positive cells)

  • Consider automated scanning platforms for consistency

  • Include calibration slides in each staining batch

  • Blind scoring by multiple trained observers

  • Establish clear criteria for positive versus negative staining

• ELISA-based quantification:

  • Generate standard curves using recombinant NTRK1

  • Perform technical and biological replicates

  • Validate antibody pairs for capture and detection

  • Optimize sample dilutions to ensure measurements within linear range

  • Account for matrix effects by using sample-matched standards

• Statistical considerations:

  • Determine appropriate sample sizes through power analysis

  • Use non-parametric tests when data do not meet normality assumptions

  • Implement appropriate multiple comparison corrections

  • Consider batch effects in longitudinal studies

  • Report both absolute values and fold changes relative to controls

• Validation strategies:

  • Confirm protein-level findings with mRNA expression data

  • Compare results across multiple detection methodologies

  • Correlate expression with functional readouts

  • Implement spike-in controls to assess recovery

  • Consider the biological context when interpreting quantitative differences