TAS1R2 Antibody, FITC conjugated

What is the optimal protocol for using TAS1R2 Antibody, FITC conjugated in immunofluorescence studies?

For optimal immunofluorescence results with TAS1R2 Antibody, FITC conjugated, follow this methodology:

• Fix cells with 4% paraformaldehyde (15-20 minutes) or tissue sections as appropriate

• Permeabilize with 0.1-0.5% Triton X-100 (10 minutes)

• Block with 5% normal serum in PBS (1 hour)

• Apply the TAS1R2 Antibody, FITC conjugated at 1:50-1:200 dilution in blocking buffer

• Incubate overnight at 4°C in a humidified chamber protected from light

• Wash 3× with PBS (5 minutes each)

• Counterstain nuclei with DAPI or propidium iodide if desired

• Mount using anti-fade mounting medium

• Image using appropriate filters (excitation/emission: 499/515 nm, laser line: 488 nm)

Remember that optimal dilutions must be determined empirically by the end user based on your specific experimental conditions .

How should TAS1R2 Antibody, FITC conjugated be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody functionality:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Protect from light exposure due to the FITC conjugate's photosensitivity

• Avoid repeated freeze/thaw cycles as this can substantially reduce antibody activity

• Store in the recommended buffer (0.01 M PBS, pH 7.4, 0.03% Proclin-300 and 50% Glycerol)

• For short-term storage (1-2 weeks), 4°C is acceptable if protected from light

• When handling the antibody, work quickly and keep the sample cold and protected from direct light

• Return to -20°C immediately after use

The concentration of the antibody solution is typically 0.25 mg/ml, and proper storage conditions are essential for maintaining this activity level .

How can TAS1R2 Antibody, FITC conjugated be used to investigate TAS1R2 dimerization with TAS1R3 in cellular models?

To investigate TAS1R2/TAS1R3 dimerization:

• Experimental approach using fluorescence techniques:

  • Co-express TAS1R2 and TAS1R3 in a suitable cell line (HEK293 recommended)

  • Use the FITC-conjugated TAS1R2 antibody alongside a distinctly labeled TAS1R3 antibody

  • Analyze receptor dimerization through advanced fluorescence techniques:

    • FRET microscopy to detect physical interaction between receptors

    • FLIM (Fluorescence Lifetime Imaging Microscopy) to quantify dimerization specifically at the plasma membrane

• Validation techniques:

  • Monitor FRET signal during photobleaching; expect exponential decrease in acceptor fluorescence with concomitant increase in donor fluorescence

  • Establish Cer/YFP fluorescence intensity relationships which should be linear for dimeric TAS1R2/TAS1R3 hetero-oligomer complexes

  • Apply sweet ligands (e.g., aspartame) to observe conformational changes in the receptor complex

• Data interpretation:

  • For wild-type TAS1R2, aspartame causes reduction in dimeric TAS1R2/TAS1R3 at the plasma membrane

  • Compare results with variant forms (e.g., TAS1R2-Val191) to examine differences in ligand response and dimerization efficiency

This approach can be used to study how mutations or ligands affect receptor dimerization and trafficking.

What are the methodological considerations when using TAS1R2 Antibody, FITC conjugated to study TAS1R2 expression in extra-oral tissues?

When investigating TAS1R2 expression in extra-oral tissues:

• Tissue-specific optimization:

  • Different tissues require modified fixation protocols (skeletal muscle: 2% PFA for 10-15 minutes; intestinal tissues: 4% PFA for 24 hours)

  • Antigen retrieval methods must be tissue-optimized (heat-induced vs. enzymatic)

  • Autofluorescence reduction is critical (e.g., use 0.1% Sudan Black B in 70% ethanol for muscle tissue)

• Controls and validation:

  • Include known TAS1R2-expressing tissues (taste buds) as positive controls

  • Use TAS1R2 knockout tissue when available as negative controls

  • Perform parallel experiments with non-conjugated TAS1R2 primary antibody + secondary detection

  • Consider comparing results with TAS1R2 mRNA expression data

• Co-localization studies:

  • Co-stain with cell-type specific markers (e.g., muscle: myosin heavy chain; intestine: villin)

  • Use Z-stack confocal microscopy to confirm subcellular localization

  • Quantify membrane vs. cytoplasmic TAS1R2 expression ratio

• Particular considerations for specific tissues:

  • For muscle tissue: examine TAS1R2 expression changes under exercise conditions

  • For intestinal tissue: investigate potential differences in TAS1R2 expression along the crypt-villus axis

  • Use normalized fluorescence intensity measurements for quantitative comparisons

How can non-specific binding be minimized when using TAS1R2 Antibody, FITC conjugated?

To minimize non-specific binding:

• Blocking optimization:

  • Test different blocking agents (5% BSA, 5-10% normal serum from the same species as secondary antibody, commercial blocking buffers)

  • Extend blocking time to 1-2 hours at room temperature

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Perform antibody titration experiments (1:50, 1:100, 1:200 dilutions)

  • Extend primary antibody incubation to overnight at 4°C

  • Prepare antibody in fresh blocking buffer

• Washing steps:

  • Increase number of washes (5-6 times instead of standard 3)

  • Extend wash duration to 10 minutes per wash

  • Add 0.05% Tween-20 to wash buffer to reduce background

• Additional techniques:

  • Pre-adsorb antibody with tissue powder from a species different from the target

  • Use appropriate isotype controls

  • Include a TAS1R2-negative tissue or TAS1R2-knockout control

• Validation controls:

  • Include secondary-only controls to assess secondary antibody background

  • If available, pre-incubate the antibody with the immunizing peptide to confirm specificity

How can TAS1R2 Antibody, FITC conjugated be used to detect conformational changes in the TAS1R2 receptor upon ligand binding?

To detect conformational changes:

• Live-cell imaging approach:

  • Transfect cells with TAS1R2 and apply the FITC-conjugated antibody to living cells to label cell-surface receptors

  • Establish baseline fluorescence parameters

  • Add sweet ligands (e.g., sucralose, neotame, acesulfame-K) at physiologically relevant concentrations

  • Monitor changes in:

    • Fluorescence distribution pattern

    • Fluorescence intensity

    • Fluorescence lifetime (if FLIM is available)

• Fixed-cell comparative studies:

  • Prepare parallel samples of cells with/without ligand exposure

  • Fix at different time points after ligand addition (0, 2, 5, 10, 30 minutes)

  • Apply TAS1R2 Antibody, FITC conjugated

  • Analyze receptor distribution, internalization patterns, and co-localization with endocytic markers

• Advanced quantitative analyses:

  • Combine with FRET techniques using TAS1R3 labeled with a FRET partner

  • Measure ligand-induced changes in fluorescence lifetime

  • Calculate receptor conformational changes using fluorescence correlation spectroscopy

  • Analyze data with specialized software to determine binding kinetics

This approach is particularly valuable for comparing wild-type TAS1R2 with variants like the Ile191Val which shows altered ligand responses.

What methodology should be used to study the impact of TAS1R2 polymorphisms on receptor function using TAS1R2 Antibody, FITC conjugated?

To study TAS1R2 polymorphisms:

• Expression system preparation:

  • Transfect HEK293 cells with plasmids expressing wild-type TAS1R2 and variant forms (e.g., Ile191Val)

  • Co-transfect with TAS1R3 to form functional heterodimers

  • Use standardized transfection protocols to ensure comparable expression levels

• Antibody-based analyses:

  • Apply TAS1R2 Antibody, FITC conjugated at standardized concentration (1:100 dilution)

  • Compare fluorescence intensities and patterns between wild-type and variant receptors

  • Quantify plasma membrane vs. cytoplasmic localization ratios

• Flow cytometry quantification:

  • Prepare parallel samples of cells expressing different TAS1R2 variants

  • Stain with TAS1R2 Antibody, FITC conjugated

  • Conduct flow cytometry to quantify:

    • Percentage of TAS1R2-expressing cells

    • Mean fluorescence intensity

    • Surface vs. total expression (with/without permeabilization)

• Functional evaluation:

  • Perform calcium imaging after ligand stimulation

  • Correlate receptor expression levels (FITC signal) with functional responses

  • Compare dose-response curves for different sweeteners (aspartame, sucralose, neotame)

• Data analysis and interpretation:

  • Use statistical methods to correlate polymorphism-specific changes in:

    • Receptor expression level

    • Membrane localization

    • Ligand-induced internalization

    • Dimerization with TAS1R3

How can TAS1R2 Antibody, FITC conjugated be used in combination with calcium imaging to study taste receptor signaling?

Integration of antibody labeling with calcium imaging:

• Sequential experimental design:

  • Step 1: Perform calcium imaging with taste stimuli

    • Load cells with calcium indicators (Fura-2/AM)

    • Record baseline [Ca²⁺]ᵢ levels

    • Apply sweet tastants (sucralose, neotame, acesulfame-K)

    • Measure calcium responses at excitation wavelengths of 340/380 nm

  • Step 2: Fix cells immediately after calcium imaging

  • Step 3: Apply TAS1R2 Antibody, FITC conjugated (1:100 dilution)

  • Step 4: Relocate the same cells and correlate receptor expression with functional responses

• Technical considerations:

  • Use gridded coverslips to relocate the same cells

  • Optimize fixation to preserve cell morphology after calcium imaging

  • Calibrate fluorescence signals to determine absolute [Ca²⁺]ᵢ concentrations

  • Apply statistical methods to correlate receptor density with response magnitude

• Advanced applications:

  • Compare wild-type vs. TAS1R2 variants (e.g., Ile191Val)

  • Study dose-dependent effects using concentration series (1-50 mM for sweeteners)

  • Analyze differences between TAS1R2 surface expression and calcium response amplitude

  • Investigate potential receptor desensitization after repeated stimulations

This combined approach allows direct correlation between receptor expression and functional responses at the single-cell level.

What techniques should be used to validate the specificity of TAS1R2 Antibody, FITC conjugated?

Comprehensive validation techniques:

• Western blot validation:

  • Run protein extracts from TAS1R2-expressing tissues/cells

  • Include recombinant TAS1R2 protein as positive control

  • Compare with TAS1R2-knockout or siRNA-treated samples as negative controls

  • Verify the detection of a single band at ~115 kDa (or multiple bands between 70-100 kDa for glycosylated/processed forms)

• Immunoprecipitation analysis:

  • Use the antibody to immunoprecipitate TAS1R2 from cell lysates

  • Confirm identity by mass spectrometry

  • Verify co-precipitation of known interacting partners (e.g., TAS1R3)

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (amino acids 183-362 of human TAS1R2)

  • Apply to parallel samples alongside non-blocked antibody

  • Confirm elimination of specific signal

• Cross-reactivity assessment:

  • Test on tissues from different species (human, rat, mouse)

  • Examine potential cross-reactivity with other TAS family members

  • Compare staining patterns with published TAS1R2 mRNA expression data

• Advanced specificity tests:

  • Conduct siRNA knockdown of TAS1R2 and verify signal reduction

  • Perform immunofluorescence on cell lines with/without TAS1R2 expression

  • Use antibodies targeting different epitopes of TAS1R2 to confirm staining pattern

How do post-translational modifications of TAS1R2 affect antibody recognition, and how can this be investigated?

Investigating post-translational modification effects:

• Analytical approach for PTM influence:

  • Treat samples with enzymes targeting specific PTMs:

    • PNGase F for N-linked glycosylation removal

    • Phosphatase for dephosphorylation

    • Deubiquitinating enzymes for ubiquitin removal

  • Compare antibody binding before/after treatments using:

    • Western blot (band shift analysis)

    • Immunofluorescence (intensity quantification)

    • Flow cytometry (signal strength measurement)

• Site-directed mutagenesis strategy:

  • Generate TAS1R2 mutants lacking specific PTM sites:

    • N-glycosylation sites (N→Q mutations)

    • Phosphorylation sites (S/T→A mutations)

  • Express wild-type and mutant receptors in cell models

  • Compare antibody binding efficiency between variants

• Specialized techniques for detailed analysis:

  • Use mass spectrometry to identify PTMs in TAS1R2 samples

  • Employ 2D gel electrophoresis to separate differently modified forms

  • Implement pulse-chase experiments to track receptor maturation and modification

  • Utilize super-resolution microscopy to examine subcellular localization differences

• Physiological relevance:

  • Compare PTM patterns between different tissues (taste buds vs. extra-oral tissues)

  • Analyze changes in PTMs upon receptor activation with sweet compounds

  • Investigate differences in PTM patterns between wild-type and variant receptors (e.g., Ile191Val)

How can TAS1R2 Antibody, FITC conjugated be applied to study metabolic disorders?

Methodological approach for metabolic research:

• Tissue-specific expression analysis:

  • Compare TAS1R2 expression in metabolically relevant tissues between healthy and diseased states:

    • Pancreatic islets (diabetes research)

    • Intestinal L-cells (incretin production)

    • Skeletal muscle (glucose utilization)

    • Adipose tissue (energy storage)

  • Quantify expression differences using standardized fluorescence intensity measurements

• Mechanistic investigations:

  • Correlate TAS1R2 expression with glucose excursions during OGTT

  • Study co-localization with metabolic signaling components:

    • Insulin receptor pathway proteins

    • GLUT transporters

    • GLP-1 in intestinal cells

  • Analyze receptor internalization in response to hyperglycemia

• Genetic variant influences:

  • Compare TAS1R2 expression patterns between individuals with different genotypes:

    • TAS1R2-Ile191Val carriers vs. wild-type

    • Other polymorphic variants

  • Correlate with clinical parameters (glucose tolerance, insulin sensitivity)

• Intervention studies:

  • Examine changes in TAS1R2 expression and localization after:

    • Exercise interventions

    • Dietary modifications

    • Pharmacological treatments

  • Correlate with improvements in metabolic parameters

This approach enables investigation of TAS1R2's role beyond taste perception in metabolic health and disease.

What is the methodology for using TAS1R2 Antibody, FITC conjugated in muscle physiology research?

Research methodology for muscle studies:

• Sample preparation techniques:

  • Fresh-frozen muscle biopsies: Snap-freeze in isopentane cooled with liquid nitrogen

  • Cryosections: 8-10 μm thickness optimal for immunofluorescence

  • Fixation: 2-4% paraformaldehyde for 10-15 minutes (excessive fixation may mask epitopes)

  • Permeabilization: 0.2% Triton X-100 for 10 minutes

  • Blocking: 5% BSA or 10% normal serum for 1 hour

• Antibody application protocol:

  • Apply TAS1R2 Antibody, FITC conjugated at 1:100 dilution

  • Incubate overnight at 4°C in humidified chamber

  • Include muscle fiber type markers (MyHC antibodies) for co-localization

  • Counterstain with DAPI for nuclear visualization

• Analysis of muscle-specific patterns:

  • Examine TAS1R2 distribution patterns:

    • Membrane vs. cytoplasmic localization

    • Fiber-type specific expression

    • Subcellular co-localization with mitochondria or sarcoplasmic reticulum

  • Compare expression between:

    • Young vs. aged muscle

    • Healthy vs. obese/diabetic subjects

    • Sedentary vs. exercise-trained individuals

• Functional correlation studies:

  • Correlate TAS1R2 expression with:

    • NAD levels (measured by enzymatic assays)

    • PARP1 activity (assessed by immunoblotting for PAR)

    • Mitochondrial function markers

    • Muscle strength and endurance parameters

This methodological approach enables investigation of TAS1R2's novel role in regulating skeletal muscle mass and function.

How should TAS1R2 Antibody, FITC conjugated be used to compare receptor expression across different species?

Cross-species comparison methodology:

• Antibody epitope evaluation:

  • Perform sequence alignment of TAS1R2 across target species

  • Focus on the antibody's target region (amino acids 183-362 in human TAS1R2)

  • Calculate sequence homology percentages to predict cross-reactivity

  • Adjust antibody concentration based on homology (higher dilutions for higher homology)

• Standardized protocol development:

  • Modify fixation parameters based on species-specific tissue properties:

    • Human samples: 4% PFA, 24 hours

    • Mouse samples: 4% PFA, 12-16 hours

    • Rat samples: 4% PFA, 16-20 hours

  • Optimize antigen retrieval for each species

  • Adjust blocking conditions to reduce species-specific background

• Validation and controls:

  • Include species-matched positive control tissues (taste buds)

  • Run parallel staining with species-specific TAS1R2 antibodies when available

  • Perform Western blots to confirm the expected molecular weight in each species

  • Include knockout/negative controls when possible

• Quantification and analysis:

  • Use identical acquisition parameters for all species

  • Apply standardized fluorescence intensity normalization

  • Employ ratio-metric analyses against internal standards

  • Present data with appropriate statistical comparisons between species

This approach allows reliable comparison of TAS1R2 expression patterns across species while accounting for technical variations.

What are the methodological considerations when comparing TAS1R2 expression in taste versus non-taste tissues using FITC conjugated antibodies?

Cross-tissue comparison methodology:

• Tissue-specific protocol optimization:

  • Taste tissue preparation:

    • Process circumvallate and foliate papillae with careful orientation

    • Use thin sections (5-7 μm) to capture individual taste buds

    • Apply mild antigen retrieval to preserve tissue architecture

  • Non-taste tissue preparation:

    • Adjust fixation times based on tissue density

    • Optimize permeabilization for each tissue type

    • Use thicker sections (10-15 μm) for intestine/muscle to capture tissue organization

• Signal normalization strategy:

  • Include internal reference markers in all tissues

  • Utilize standardized exposure settings across all samples

  • Apply background subtraction tailored to each tissue type

  • Consider autofluorescence correction methods for naturally fluorescent tissues

• Comprehensive controls:

  • Process sequential sections for H&E staining to confirm tissue morphology

  • Include parallel sections for RNA in situ hybridization to correlate protein with mRNA

  • Run sections with secondary antibody only for each tissue type

  • When possible, include tissues from TAS1R2-deficient organisms

• Advanced analytical approaches:

  • Employ laser scanning confocal microscopy for subcellular localization

  • Implement spectral unmixing for tissues with high autofluorescence

  • Quantify receptor density using standardized ROI measurements

  • Analyze co-localization with tissue-specific markers using Pearson's correlation coefficient