TAS1R3 Antibody

What criteria should researchers consider when selecting a TAS1R3 antibody?

When selecting a TAS1R3 antibody for research, several critical factors must be evaluated systematically:

• Target epitope specificity: Consider whether you need antibodies targeting specific regions such as the extracellular domain (amino acids 431-530, 400-570, or 508-521), internal regions, or C-terminal regions. Each region may provide different biological insights .

• Host species compatibility: Select an antibody raised in a species distinct from your experimental tissue to avoid cross-reactivity. Rabbit-derived polyclonal antibodies are most commonly available for TAS1R3 .

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, ELISA, IHC, IF/ICC). Not all antibodies perform equally across different techniques .

• Species reactivity: Confirm the antibody reacts with your species of interest. Most commercial TAS1R3 antibodies react with human samples, while fewer are validated for mouse and rat samples .

• Clonality considerations: Most available TAS1R3 antibodies are polyclonal, offering broader epitope recognition but potentially more batch-to-batch variability .

How should researchers validate a TAS1R3 antibody before experimental use?

Proper antibody validation is essential to ensure reliable experimental results. A systematic validation approach should include:

• Positive and negative control tissues: Test the antibody on tissues known to express TAS1R3 (positive controls like taste buds, hypothalamus, or cell lines like K-562, THP-1) and tissues with minimal expression (negative controls) .

• Blocking peptide experiments: Use the corresponding blocking peptide to confirm specificity. Signal abrogation in the presence of the blocking peptide indicates specificity, as demonstrated in Western blot analyses of brain lysates .

• Validation across techniques: Confirm antibody performance in multiple techniques (e.g., if planning both Western blot and immunohistochemistry experiments) .

• Expression correlation: Compare protein detection with mRNA expression data if available .

• Transfection studies: Test antibody specificity using cells transfected with TAS1R3 versus non-transfected cells. This approach was effectively used to validate TAS1R3 antibodies against related receptors like TAS1R1-2 and mGlu receptors .

What are the optimal protocols for using TAS1R3 antibodies in Western blot analysis?

For optimal Western blot results with TAS1R3 antibodies:

• Sample preparation:

  • Use fresh tissue lysates with protease inhibitors to prevent degradation

  • For membrane proteins like TAS1R3, include mild detergents in lysis buffers

  • Load 25μg protein per lane as a starting point

• Dilution optimization:

  • Begin with the manufacturer's recommended range (typically 1:500-1:1000)

  • Perform a dilution series if signal-to-noise ratio is suboptimal

• Secondary antibody selection:

  • For rabbit-derived primaries, HRP-conjugated goat anti-rabbit IgG has shown good results at 1:10000 dilution

• Blocking conditions:

  • 3% nonfat dry milk in TBST has been successfully used with TAS1R3 antibodies

• Detection sensitivity:

  • Use ECL detection systems, with exposure times ranging from 1-5 seconds for standard applications

• Expected molecular weight: Look for bands around 93 kDa, which is the calculated molecular weight of TAS1R3

What methodological considerations apply when using TAS1R3 antibodies for immunohistochemistry?

When performing immunohistochemistry with TAS1R3 antibodies:

• Tissue preparation options:

  • For frozen sections: Use 4% paraformaldehyde fixation followed by cryoprotection in 20% sucrose

  • For paraffin sections: Optimize antigen retrieval methods, as TAS1R3 epitopes may be sensitive to masking

• Blocking solution composition:

  • Use 3% BSA, 0.3% Triton X-100, 2% serum matching secondary antibody species, and 0.1% sodium azide in PBS

• Primary antibody incubation:

  • Typical dilutions range from 1:100-1:500

  • Optimal incubation is 16 hours at 4°C for most protocols

• Controls required:

  • Include peptide competition controls using the immunizing peptide (e.g., 10μM concentration)

  • Use preimmune serum controls to assess background

  • Include known positive tissue controls such as taste papillae or hypothalamus

• Signal detection systems:

  • For fluorescence: Cy3-conjugated or Alexa Fluor-conjugated secondary antibodies work well

  • For chromogenic detection: HRP/DAB systems provide permanent staining

How can researchers effectively use TAS1R3 antibodies to study receptor heterodimerization?

Studying TAS1R3 heterodimerization requires specialized approaches:

• Co-immunoprecipitation (co-IP) protocol:

  • Validate antibody specificity against potential heterodimer partners (e.g., TAS1R1, TAS1R2, mGlu2)

  • Use crosslinking agents to stabilize weak interactions

  • Include appropriate negative controls (IgG, unrelated receptors)

  • Western blot analysis of immunoprecipitates to confirm interaction

• Bioluminescence Resonance Energy Transfer (BRET):

  • For recombinant systems, use validated cell models like HEK-293

  • Design constructs with appropriate tags for energy transfer experiments

  • Control experiments should include individual receptor expressions

  • Analyze concentration-response relationships of heterodimerization

• Co-localization analysis:

  • Use dual-label immunocytochemistry with antibodies recognizing different receptor types

  • Quantify co-localization using appropriate algorithms

  • Ensure antibodies are raised in different host species or directly conjugated to different fluorophores

Data table from research on mGlu2/TAS1R3 heterodimerization:

Data adapted from heterodimerization studies with pertussis toxin (PTX) sensitivity

What approaches can be used to assess TAS1R3 expression patterns across different tissues?

To comprehensively evaluate TAS1R3 expression patterns:

• Multi-technique validation approach:

  • Combine antibody-based protein detection with mRNA analysis

  • Compare RT-qPCR, RNA-seq, and protein detection methods

  • Use multiple antibodies targeting different epitopes when possible

• Single-cell analysis protocols:

  • Flow cytometry: For cell surface expression, use live intact cells and non-permeabilizing conditions

  • Single-cell RNA-seq complemented with immunocytochemistry

  • Avoid fixation artifacts by testing multiple fixation protocols

• Tissue-specific considerations:

  • Brain tissue: Use perfusion fixation to preserve antigenicity

  • Blood leukocytes: Analyze fresh isolated cells rather than fixed samples

  • Taste tissue: Microdissect specific papillae types for region-specific analysis

Research findings show TAS1R3 is highly expressed in multiple tissues beyond taste buds:

Data compiled from expression studies across multiple tissues

How can researchers address common issues when using TAS1R3 antibodies in experiments?

When encountering problems with TAS1R3 antibodies, systematic troubleshooting is essential:

• High background in immunostaining:

  • Increase blocking time and concentration (use 5% serum instead of 2%)

  • Test different detergent concentrations (reduce Triton X-100 from 0.3% to 0.1%)

  • Use species-specific blocking reagents to reduce non-specific binding

  • Include additional washing steps with higher salt concentrations

• Multiple bands in Western blot:

  • Verify if bands represent glycosylation variants (treat samples with glycosidases)

  • Test if bands represent degradation products (add more protease inhibitors)

  • Compare with positive control tissues known to express TAS1R3

  • Use peptide competition to identify specific bands

  • Consider using antibodies targeting different epitopes for confirmation

• Weak or no signal:

  • For Western blot: Increase protein loading (50μg instead of 25μg)

  • For IHC/ICC: Optimize antigen retrieval (test multiple methods)

  • Test shorter fixation times to preserve epitope accessibility

  • Increase antibody concentration or incubation time

  • Try signal amplification systems (TSA, ABC method)

• Inconsistent results between experiments:

  • Use consistent lot numbers of antibodies when possible

  • Prepare larger aliquots of working solutions

  • Include standardized positive controls in each experiment

  • Document detailed protocols including all reagent sources

What approaches can resolve contradictory findings when using different TAS1R3 antibodies?

When different TAS1R3 antibodies yield contradictory results:

• Systematic comparison strategy:

  • Test all antibodies side-by-side under identical conditions

  • Compare antibodies targeting different epitopes of TAS1R3

  • Validate each antibody using knockout/knockdown controls if available

  • Correlate findings with mRNA expression data

• Epitope accessibility considerations:

  • Different fixation methods may affect epitope accessibility differently

  • Membrane topology may mask certain epitopes in native conditions

  • Protein-protein interactions may block antibody binding sites

• Cross-reactivity analysis:

  • Test for cross-reactivity with related receptors (TAS1R1, TAS1R2, mGlu receptors)

  • Use heterologous expression systems to assess specificity

  • Perform peptide competition with both specific and related peptides

• Integration of multiple approaches:

  • Combine antibody-based techniques with genetic approaches

  • Use CRISPR-mediated tagging to validate antibody binding

  • Apply proximity ligation assays for increased specificity

How can TAS1R3 antibodies be applied to study non-canonical functions of taste receptors?

Investigating non-canonical functions of TAS1R3 requires specialized approaches:

• Immune cell function studies:

  • Use validated TAS1R3 antibodies to sort receptor-positive immune subpopulations

  • Apply blocking antibodies to assess receptor function in immune responses

  • Combine with cytokine assays to measure functional outcomes

• Chemosensory GPCR heterodimerization:

  • Investigate TAS1R3 interactions with non-taste GPCRs

  • Apply proximity-based assays (BRET, FRET, PLA) in native tissues

  • Use antibodies targeting different epitopes to avoid steric hindrance

• Metabolic regulation investigations:

  • Study TAS1R3 in metabolic tissues using co-staining with metabolic markers

  • Apply receptor-specific antagonists in combination with immunocytochemistry

  • Correlate receptor expression with metabolic parameters

Research has revealed TAS1R3's role beyond taste perception:

Data derived from studies of non-canonical TAS1R3 functions

What considerations apply when using TAS1R3 antibodies to study polymorphic variants of the receptor?

When investigating TAS1R3 polymorphic variants:

• Epitope sequence verification:

  • Confirm whether the antibody's target epitope contains polymorphic sites

  • Review sequence alignment of variants to assess potential antibody binding differences

  • Consider raising custom antibodies against variant-specific sequences

• Expression level quantification:

  • Use quantitative Western blot techniques with recombinant protein standards

  • Apply flow cytometry for cell surface expression quantification

  • Correlate protein levels with mRNA expression of specific variants

• Functional correlation approaches:

  • Combine genotyping with antibody-based protein detection

  • Correlate receptor abundance with functional phenotypes

  • Use heterologous expression systems to compare variant proteins

Research on TAS1R3 polymorphisms has identified functional variants:

Data on TAS1R3 polymorphisms associated with taste preferences

How do different applications of TAS1R3 antibodies compare in terms of sensitivity and specificity?

Different experimental applications have distinct performance characteristics:

Data compiled from multiple sources on application-specific considerations

What are the advanced approaches for quantitative analysis of TAS1R3 using antibody-based methods?

For rigorous quantitative analysis of TAS1R3:

• Absolute quantification methods:

  • Use purified recombinant TAS1R3 protein standards in Western blots

  • Apply AQUA peptide approaches with mass spectrometry

  • Include calibration controls in flow cytometry experiments

• Relative quantification approaches:

  • Normalize to appropriate housekeeping proteins for Western blot

  • Use ratiometric imaging in immunofluorescence studies

  • Apply digital droplet PCR combined with protein quantification

• Dynamic expression analysis:

  • Time-course experiments with synchronized sampling

  • Live-cell imaging with compatible antibody fragments

  • Inducible expression systems with antibody validation

• Spatial distribution quantification:

  • High-content imaging with automated analysis

  • Tissue microarray approaches for comparative studies

  • Laser capture microdissection combined with protein extraction