PTH1R Antibody, FITC conjugated

What is PTH1R and what is its biological function?

PTH1R (Parathyroid Hormone Receptor 1) functions as a receptor for both parathyroid hormone and parathyroid hormone-related peptide. This receptor plays a crucial role in cellular signaling through its interaction with G proteins that activate adenylyl cyclase and the phosphatidylinositol-calcium second messenger system . PTH1R is also known by several aliases including PTHR, PTHR1, PTH/PTHrP type I receptor, and PTH/PTHr receptor . The receptor is particularly important in bone metabolism, where it mediates signaling primarily through G protein pathways . Understanding the fundamental biology of this receptor is essential for interpreting experimental results when using antibodies targeted against it.

What applications are validated for PTH1R Antibody, FITC conjugated?

The PTH1R Antibody, FITC conjugated has been validated primarily for ELISA applications according to manufacturer specifications . Western blot (WB) applications are also mentioned for non-conjugated versions of PTH1R antibodies with recommended dilutions of 1:500-1:1000 . Immunohistochemistry (IHC) applications are also suitable for some PTH1R antibodies with recommended dilutions of 1:50-1:500 . When designing experiments, researchers should consider that the FITC conjugation makes this antibody particularly valuable for flow cytometry and fluorescence microscopy applications where direct detection without secondary antibodies is advantageous.

What are the recommended storage and handling conditions?

For optimal performance and longevity, the PTH1R Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . Researchers should avoid repeated freeze-thaw cycles as this can degrade the antibody and reduce its effectiveness. The antibody is provided in a liquid form with a buffer composition of 50% Glycerol, 0.01M PBS, pH 7.4, and contains 0.03% Proclin 300 as a preservative . When handling the antibody, it's advisable to aliquot it into smaller volumes before freezing to minimize freeze-thaw cycles during experimental use.

What species reactivity has been confirmed for this antibody?

The PTH1R Antibody, FITC conjugated has been confirmed to react with human samples . Some non-conjugated versions of PTH1R antibodies have shown reactivity with mouse and rat samples as well . When testing the antibody in a new experimental system, researchers should first validate its reactivity in their specific model organism, particularly if working with species other than human. Testing antibody specificity using positive and negative controls is essential before proceeding with critical experiments.

What is the immunogen used for generating this antibody?

The PTH1R Antibody, FITC conjugated is developed using recombinant Human Parathyroid hormone/parathyroid hormone-related peptide receptor protein (specifically amino acids 485-593) . Understanding the immunogen is critical for interpreting potential cross-reactivity and binding characteristics. This antibody is a polyclonal IgG developed in rabbit , which influences its binding characteristics and applications in various experimental settings.

How can PTH1R Antibody, FITC conjugated be used to investigate signaling bias in GPCR pathways?

Recent research has revealed that PTH1R signaling can demonstrate biased signaling between G protein and β-arrestin pathways. A study identified an antibody (ECD-scFvhFc) that acts as a β-arrestin 2 antagonist while allowing canonical G protein signaling to proceed . To investigate signaling bias using FITC-conjugated PTH1R antibodies, researchers can implement the following methodology:

• Establish cell models expressing PTH1R with downstream pathway reporters for both G protein (cAMP) and β-arrestin (recruitment assays) signaling.

• Pre-treat cells with the FITC-conjugated PTH1R antibody at varying concentrations.

• Challenge with PTH or PTHrP ligands and measure the differential activation of signaling pathways.

• Analyze the impact of antibody binding on receptor conformation using techniques such as FRET or BRET between the FITC label and other fluorescent tags on signaling components.

This approach allows researchers to determine whether antibody binding to the extracellular domain (ECD) influences the conformational changes required for selective pathway activation, similar to the biased signaling observed with ECD-binding antibodies in previous studies .

What are the optimal methods for using PTH1R Antibody, FITC conjugated in flow cytometry applications?

For optimal flow cytometry results with PTH1R Antibody, FITC conjugated, researchers should follow this methodological approach:

• Cell preparation: Harvest cells expressing PTH1R (either endogenous or recombinant) and wash in PBS containing 1-2% serum or BSA to block non-specific binding.

• Antibody concentration optimization: Titrate the antibody (starting with manufacturer recommendations) to determine the optimal concentration that provides the best signal-to-noise ratio. Based on similar antibodies, concentrations ranging from 1:50 to 1:500 may be appropriate .

• Controls: Include appropriate controls:

  • Isotype control (FITC-conjugated rabbit IgG)

  • Negative cell population (cells not expressing PTH1R)

  • Blocking control (pre-incubation with excess unlabeled PTH1-34 to compete for receptor binding)

• Gating strategy: When analyzing data, implement a hierarchical gating strategy:

  • Gate on single cells (FSC-H vs FSC-A)

  • Gate on viable cells (using a viability dye)

  • Analyze FITC signal intensity in the appropriate detection channel

This approach will help differentiate specific binding from autofluorescence and non-specific binding, providing reliable quantification of PTH1R expression levels on target cells.

How can researchers validate the specificity of PTH1R Antibody binding in experimental systems?

Validating antibody specificity is critical for generating reliable research data. For PTH1R Antibody, FITC conjugated, implement these validation strategies:

• Competition assay: Pre-incubate the antibody with an excess of PTH 1-34 peptide. Specific binding should be significantly reduced, as demonstrated in previous research where "the specificity of the fusion protein binding to PTHR1 was determined by its competition with an excess of PTH 1–34" .

• Knockout/knockdown controls: Compare antibody staining between wild-type cells and those with CRISPR/Cas9 knockout or siRNA knockdown of PTH1R.

• Positive control cell lines: Use cell lines known to express PTH1R, such as osteoblastic HOS cells or the various cell lines tested in Western blot applications (Jurkat, MCF7, U251, DU145, THP1) .

• Recombinant expression system: Compare staining in cells transfected with PTH1R expression construct versus empty vector control.

• Cross-validation with multiple antibodies: Compare staining patterns with other validated anti-PTH1R antibodies targeting different epitopes.

This multi-faceted validation approach ensures that the observed signals truly represent PTH1R localization and not non-specific binding or artifacts.

What techniques can be used to study PTH1R-ligand interactions using FITC-conjugated antibodies?

To investigate PTH1R-ligand interactions using FITC-conjugated antibodies, researchers can implement several sophisticated techniques:

• Competitive binding assays: Use increasing concentrations of unlabeled ligands (PTH or PTHrP) to compete with the FITC-conjugated antibody binding. Analysis using flow cytometry or plate-based fluorescence detection can generate binding curves and determine relative binding affinities. Previous research has established that PTH1R ECD binds to PTH (1-34) with a KD of approximately 4 μM .

• FRET-based interaction studies: Develop an experimental system where the FITC-conjugated antibody acts as a donor fluorophore and a receptor-bound ligand labeled with an appropriate acceptor fluorophore enables energy transfer measurements upon binding.

• Time-resolved binding kinetics: Using real-time confocal microscopy, monitor the association and dissociation rates of FITC-conjugated antibodies to cell surface PTH1R in the presence or absence of receptor ligands.

• Receptor internalization studies: Monitor changes in surface versus internalized FITC fluorescence following ligand stimulation to assess how different ligands modify receptor trafficking.

These approaches can provide valuable insights into how different ligands and conditions affect PTH1R binding properties and subsequent cellular responses.

How can PTH1R Antibody, FITC conjugated be used alongside other tools to study receptor dynamics?

Combining FITC-conjugated PTH1R antibodies with complementary research tools creates powerful experimental systems for studying receptor dynamics:

• Multi-color imaging: Combine the FITC-conjugated PTH1R antibody with other spectrally distinct fluorophores to simultaneously visualize:

  • PTH1R (FITC channel)

  • G protein subunits (e.g., Gαs tagged with RFP)

  • β-arrestin (tagged with far-red fluorophores)

  • Clathrin or caveolin (to study endocytic mechanisms)

• Integration with fusion protein approaches: Building on research that developed PTH-HRP and PTH-APEX2 fusion proteins , researchers can combine these tools with FITC-antibody detection to correlate ligand binding with receptor localization.

• Pulse-chase experiments: Use the FITC-conjugated antibody to label the initial pool of surface receptors, then track their fate following various stimulation conditions.

• Super-resolution microscopy: The FITC fluorophore is compatible with techniques like STORM and PALM, enabling nanoscale resolution of receptor organization in the plasma membrane.

• Live-cell antibody feeding assays: Add the FITC-conjugated antibody to live cells to track the fate of newly delivered receptors, distinguishing this population from the total receptor pool.

These integrated approaches provide a comprehensive view of PTH1R dynamics, from initial ligand binding through signaling activation and receptor trafficking.

What factors might affect the performance of PTH1R Antibody, FITC conjugated?

Several technical factors can influence antibody performance and should be considered when optimizing experimental protocols:

• Buffer compatibility: The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Ensure experimental buffers are compatible and maintain proper pH to preserve antibody function.

• Photobleaching: FITC is susceptible to photobleaching. Minimize exposure to light during storage and experimental procedures, and consider using anti-fade reagents for microscopy applications.

• pH sensitivity: FITC fluorescence is optimal at alkaline pH (8.0-9.0) and decreases at acidic pH. This is particularly relevant when tracking internalized receptors into endosomal/lysosomal compartments where pH is acidic.

• Fixation protocols: If using fixed cells or tissues, optimize fixation methods, as excessive fixation can damage epitopes and reduce antibody binding. Different fixatives (paraformaldehyde, methanol, etc.) may affect FITC fluorescence differently.

• Autofluorescence: Certain cells and tissues exhibit natural autofluorescence in the FITC channel. Appropriate controls and background subtraction are essential for accurate interpretation.

By addressing these factors proactively, researchers can optimize experimental conditions and obtain more reliable and reproducible results.

How can researchers quantify PTH1R expression levels using FITC-conjugated antibodies?

Accurate quantification of PTH1R expression using FITC-conjugated antibodies requires rigorous methodological approaches:

• Flow cytometry quantification:

  • Use calibration beads with known quantities of FITC molecules to establish a standard curve

  • Convert mean fluorescence intensity (MFI) to molecules of equivalent soluble fluorochrome (MESF)

  • Compare test samples to this standard for approximate receptor density

• Microscopy-based quantification:

  • Implement standardized image acquisition settings

  • Use internal controls with known expression levels

  • Apply consistent thresholding algorithms

  • Quantify total fluorescence intensity per cell or membrane-specific fluorescence

• Plate-based fluorometry:

  • Establish standard curves with recombinant PTH1R

  • Implement consistent cell numbers per well

  • Account for cell size differences between samples

• Western blot correlation:

  • When possible, correlate FITC signal intensity from flow cytometry or microscopy with Western blot quantification using non-conjugated PTH1R antibodies at 1:500-1:1000 dilution

  • Use internal loading controls for normalization

These quantitative approaches enable comparative studies across different experimental conditions, cell types, or disease states.

How can PTH1R Antibody, FITC conjugated contribute to drug discovery research?

The PTH1R Antibody, FITC conjugated can serve as a valuable tool in drug discovery applications through several methodological approaches:

• High-throughput screening assays:

  • Develop flow cytometry or plate-based fluorescence assays to screen compounds that compete with antibody binding

  • This approach builds on previous research demonstrating that "bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding"

• Receptor conformational dynamics:

  • Utilize FRET between the FITC-conjugated antibody and other fluorescently labeled domains to detect ligand-induced conformational changes

  • This approach can identify compounds that induce specific receptor conformations associated with biased signaling

• Antibody epitope mapping:

  • Map the binding epitope of the PTH1R Antibody (which targets amino acids 485-593 of the receptor)

  • Compare with known binding sites of therapeutic peptides to identify potential overlapping binding regions

• Receptor trafficking modulators:

  • Screen for compounds that alter receptor internalization, recycling, or degradation using the FITC-conjugated antibody to track receptor localization

• Receptor expression profiling:

  • Quantify receptor expression across diverse tissue samples to identify target tissues with high receptor expression for drug development

These approaches can accelerate the discovery of novel therapeutics targeting PTH1R for bone disorders and other related conditions.

What are the considerations for using PTH1R Antibody, FITC conjugated in co-localization studies?

For co-localization studies investigating PTH1R interactions with other cellular components, researchers should implement these methodological considerations:

• Spectral compatibility:

  • FITC excitation/emission (approximately 495/519 nm) must be spectrally separated from other fluorophores to avoid bleed-through

  • Compatible fluorophores include:

    • DAPI or Hoechst for nuclear staining

    • RFP, Texas Red, or rhodamine for second targets

    • Far-red fluorophores like Cy5 for third targets

• Sequential imaging:

  • Acquire FITC channel separately from channels with potential spectral overlap

  • Apply appropriate compensation algorithms during analysis

• Controls for co-localization analysis:

  • Positive control: Known interacting proteins

  • Negative control: Proteins that should not co-localize

  • Single-color controls to determine bleed-through

• Quantitative co-localization metrics:

  • Calculate Pearson's correlation coefficient, Mander's overlap coefficient, or other appropriate metrics

  • Apply threshold values consistently across experimental conditions

• Super-resolution considerations:

  • For higher resolution co-localization, consider techniques compatible with FITC such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy

These approaches enable rigorous investigation of PTH1R interactions with signaling partners, scaffolding proteins, or other components of the cellular machinery.