TACR1 Antibody

What is TACR1 and why is it a significant research target?

TACR1 (Tachykinin Receptor 1), also known as Neurokinin-1 receptor (NK1R) or Substance P receptor (SPR), is a G protein-coupled receptor consisting of 407 amino acid residues with a molecular weight of approximately 46-58 kDa . It contains seven hydrophobic transmembrane domains with three extracellular and three intracellular loops, an amino-terminus, and a cytoplasmic carboxy-terminus .

The receptor is significant in research because:

• It mediates phosphatidylinositol metabolism through the tachykinin substance P

• It's involved in the transmission of stress signals, pain, smooth muscle contraction, and inflammation

• It plays roles in depression, anxiety, stress, behavioral responses, neuronal survival, and cardiovascular/respiratory functions

• It's implicated in multiple disease pathways including rheumatoid arthritis, osteoarthritis, asthma, and gastrointestinal disorders

• TACR1 antagonists such as aprepitant have therapeutic potential in neurological and psychiatric conditions

What are the key considerations when selecting a TACR1 antibody for my research?

When selecting a TACR1 antibody, consider these critical parameters:

Select antibodies with robust validation data for your specific application and model organism .

What are the optimal protocols for using TACR1 antibodies in Western blotting?

For optimal Western blot detection of TACR1:

Sample Preparation:

• Extract proteins from tissues/cell lines expressing TACR1 (e.g., brain tissue, SH-SY5Y, THP-1, IMR5, SK-N-BE, Kelly cells)

• Load 25-30 μg protein per lane (as validated in published protocols)

Protocol Optimization:

• Primary antibody dilution: 1:500-1:2000 (antibody-dependent)

• Expected molecular weight detection: 45-55 kDa (may vary slightly depending on post-translational modifications)

• Blocking recommendation: 3% nonfat dry milk in TBST

• Secondary antibody: HRP-conjugated anti-host IgG at 1:10,000 dilution

Validation Controls:

• Positive controls: Neuroblastoma cell lines (IMR5, SK-N-BE, Kelly)

• Negative controls: Fibroblasts (minimal signal expected)

• Specificity verification: Competitive inhibition with immunizing peptide

How can I optimize immunocytochemistry/immunofluorescence protocols for TACR1 localization studies?

For successful TACR1 subcellular localization:

Sample Preparation:

• Fix cells using 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.2% Triton X-100 (10 minutes)

• Block with 5% normal serum from the secondary antibody host species

Staining Protocol:

• Primary antibody dilutions: 1:50-1:200 for ICC/IF applications

• Incubate overnight at 4°C or 2 hours at room temperature

• Secondary antibody: Fluorophore-conjugated anti-host IgG (1:200-1:500)

• Counterstain nuclei with DAPI

Technical Considerations:

• For membrane receptor visualization, reduce permeabilization time

• For double-labeling with neuronal markers, select antibodies raised in different host species

• Use TACR1-Atto 488 directly conjugated antibodies to eliminate secondary antibody steps

• Image using confocal microscopy for precise subcellular localization

How can TACR1 antibodies be used to study receptor-ligand interactions and signaling pathways?

Co-immunoprecipitation Studies:

• Use anti-TACR1 antibodies to pull down receptor complexes following substance P stimulation

• Analyze co-precipitated proteins by Western blot or mass spectrometry to identify signaling partners

• Compare signaling complex formation between control and substance P-stimulated conditions

Phosphorylation State Analysis:

• Stimulate cells with substance P (100-500 nM) for various timepoints (5-60 min)

• Immunoprecipitate TACR1 using validated antibodies

• Probe with phospho-specific antibodies (e.g., anti-phospho-SRC) to detect activation of downstream pathways

• Quantify relative phosphorylation levels normalized to total TACR1

Competitive Binding Assays:

• Pre-incubate cells with substance P (100-500 nM) before TACR1 antagonist treatment (e.g., fosaprepitant)

• Use TACR1 antibodies to assess receptor internalization by comparing membrane vs. cytoplasmic fractions

• Quantify changes in downstream effectors like E2F2 and TP53 pathway components

What are the approaches for validating TACR1 antibody specificity in neurological tissue samples?

Multi-method Validation Approach:

Specialized Neurological Validation:

• Use brain region-specific positive controls (amygdala, hypothalamus, nucleus accumbens)

• Perform co-localization studies with established neuronal markers

• Compare staining patterns between human, rat, and mouse brain sections (accounting for species differences)

How should I address discrepancies in TACR1 molecular weight detection across different experimental systems?

TACR1 antibodies typically detect bands between 45-58 kDa, but variations occur due to:

Common Causes of Molecular Weight Variations:

• Post-translational modifications (glycosylation, phosphorylation)

• Presence of multiple isoforms (NP_001049.1 and NP_056542.1)

• Tissue/cell-specific processing

Methodological Approach to Discrepancy Resolution:

• Employ phosphatase or glycosidase treatments to determine contribution of modifications

• Compare detection patterns across antibodies targeting different epitopes

• Incorporate isoform-specific positive controls

• Use more stringent denaturing conditions to eliminate aggregation artifacts

Interpretation Framework:

• 46 kDa: Theoretical unmodified molecular weight

• 55-58 kDa: Commonly observed in brain tissue (likely glycosylated forms)

• 38-40 kDa: May represent truncated or alternative splice variants

What strategies can resolve non-specific binding and high background issues when using TACR1 antibodies?

Systematic Troubleshooting Approach:

Validation Controls:

• Include peptide competition controls to distinguish specific from non-specific signals

• Use TACR1-knockout or knockdown samples as negative controls

• Compare commercially validated cell lines (SH-SY5Y, THP-1, Caco-2, HeLa)

How can TACR1 antibodies be leveraged for studying neuroblastoma and other cancer models?

Recent research demonstrates TACR1's significance in neuroblastoma :

Experimental Approaches:

• Cell Viability Assessment:

  • Treat neuroblastoma cell lines (IMR5, SK-N-BE, Kelly) with TACR1 antagonists

  • Measure viability using MTT assays before and after treatment

  • Assess dose-response relationships with TACR1 antagonists like aprepitant

• Pathway Analysis:

  • Use TACR1 antibodies to monitor receptor expression levels across different neuroblastoma cell lines

  • Correlate TACR1 expression with SRC phosphorylation status

  • Measure changes in downstream targets (E2F2, TP53) following receptor inhibition

• In Vivo Applications:

  • Monitor tumor TACR1 expression in xenograft models

  • Correlate expression with tumor progression and treatment response

  • Use fluorescently-labeled TACR1 antibodies for in vivo imaging studies

Research Findings:

• High TACR1 expression correlates with increased p-SRC in aggressive neuroblastoma cell lines

• TACR1 inhibition reduces cell viability and induces apoptosis in neuroblastoma cells

• TACR1 antagonism represses E2F2 and induces TP53 signaling

• In vivo TACR1 inhibition with aprepitant reduces neuroblastoma xenograft tumor burden

What are the current methodological advances in using TACR1 antibodies for studying neuroinflammatory conditions?

Advanced Techniques:

• Multi-label Immunohistochemistry:

  • Combine TACR1 antibodies with markers for specific immune cell populations (microglia, astrocytes)

  • Use multiplexed antibody panels to simultaneously detect TACR1, substance P, and inflammatory cytokines

  • Apply tissue clearing techniques for 3D visualization of TACR1 distribution in intact brain structures

• Single-cell Analysis:

  • Sort TACR1-positive cells using antibody-based flow cytometry

  • Analyze transcriptomes of isolated populations

  • Correlate TACR1 expression levels with inflammatory gene signatures

• In Situ Proximity Ligation Assay (PLA):

  • Detect TACR1 interactions with inflammatory signaling components

  • Visualize receptor complexes at subcellular resolution

  • Quantify interaction changes during disease progression

Clinical Application Strategy:

• Use validated TACR1 antibodies on patient-derived samples from neuroinflammatory conditions

• Correlate TACR1 expression patterns with disease severity and treatment response

• Develop tissue-based diagnostic approaches for stratifying patients for TACR1-targeted therapies