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| Targets |
This probe targets the parathyroid hormone 1 receptor (PTH1R), a class B G protein-coupled receptor (GPCR) that regulates calcium and phosphate homeostasis primarily in bone and kidney. The biotinylated peptide retains high affinity (Kd in the low nM range) for the PTH1R, as the biotin is attached at a site that does not disrupt the core binding interface (residues 1-34). Upon binding to PTH1R, the PTH(1-34) fragment activates Gs and Gq signaling pathways, leading to increased cAMP production and intracellular calcium mobilization.
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| ln Vitro |
In radioligand competition binding assays using membranes from cells expressing human PTH1R (e.g., SaOS-2 osteosarcoma cells, HEK293-PTH1R), unlabeled biotinylated PTH(1-34) competes with ¹2⁵I-labeled [Nle⁸,¹⁸,Tyr3⁴]PTH(1-34) with an IC50 in the low nanomolar range (typically 1-5 nM), confirming that biotinylation does not abolish receptor binding. In cell-based functional assays (cAMP accumulation, CRE-luciferase reporter, or calcium flux), biotin-PTH(1-34) stimulates PTH1R with similar potency (EC50 ~1-10 nM) as the unlabeled peptide, demonstrating full agonist activity.
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| ln Vivo |
In vivo, biotin-PTH(1-34) is seldom administered to animals due to the biotin label potentially altering pharmacokinetics and the availability of non-labeled peptides. Instead, it is used ex vivo (in tissue sections) and in vitro. For receptor binding analysis, 5-10 ug of biotin-PTH(1-34) is used to label PTH1R in cryosections of kidney or bone tissue, followed by detection with fluorescently labeled streptavidin (e.g., Alexa Fluor 488) for histochemical mapping of receptor distribution.
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| Enzyme Assay |
A 96-well plate is coated with 5 ug/mL neutravidin (in PBS) overnight at 4degC, then blocked with 1% BSA in PBS for 1 hour at room temperature. Biotin-PTH(1-34) (0.1-10 nM in PBS + 0.1% BSA) is added to the wells and incubated for 30-60 minutes. After washing, PTH1R-containing membrane preparations (2-10 ug protein per well in binding buffer: 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 0.1% BSA) are added and incubated for 2 hours at room temperature. Bound receptor is detected using an anti-PTH1R antibody followed by HRP-conjugated secondary antibody and TMB substrate. For competition assays, increasing concentrations (0.1-100 nM) of test compounds (e.g., unlabeled PTH or antagonists) are added together with the membrane preparation. Alternatively, for determining binding kinetics via surface plasmon resonance (SPR), biotin-PTH(1-34) is immobilized on a streptavidin-coated sensor chip, and PTH1R analyte is flowed over the chip. Association (kon) and dissociation (koff) rates are determined. The dissociation constant (Kd) is calculated by kinetic fitting.
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| Cell Assay |
HEK293 cells stably expressing human PTH1R (HEK293-PTH1R) are seeded in 96-well white-walled plates (3×10⁴ cells/well) in DMEM + 10% FBS and incubated overnight. Cells are washed with HBSS + 0.1% BSA + 0.5 mM IBMX (phosphodiesterase inhibitor). Varying concentrations (0.001-1000 nM) of biotin-PTH(1-34) diluted in HBSS/BSA/IBMX are added to the wells and incubated for 15 min at 37degC. The reaction is terminated by adding 0.1 M HCl, and the plate is shaken for 10 min. Intracellular cAMP is measured using a competitive ELISA or HTRF kit (Cisbio) following the manufacturer's instructions. The EC50 value is determined using a four-parameter logistic curve. For live-cell imaging, HEK293-PTH1R or SaOS-2 cells are seeded in 8-well chamber slides or 96-well optical plates. Cells are incubated with 10-100 nM biotin-PTH(1-34) (diluted in phenol red-free DMEM + 0.1% BSA) for 30 min at 4degC (to prevent internalization). After washing, cells are incubated with Alexa Fluor 488- or 555-labeled streptavidin (2 ug/mL) for 20 min at 4degC, washed, and immediately imaged by confocal microscopy. To study PTH1R internalization and trafficking, the temperature is shifted to 37degC after ligand binding, and internalization is tracked at 0, 5, 15, 30, and 60 minutes. Cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and counterstained with DAPI. Receptor-ligand co-localization is analyzed by confocal microscopy. Use of fluorescent streptavidin (Alexa Fluor 488, 555) with different emission spectra enables multiplexing with other fluorophores.
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| Animal Protocol |
For ex vivo receptor autoradiography, tissues (e.g., mouse kidney, femur) are snap-frozen, sectioned (10-20 um) using a cryostat, and thaw-mounted onto Superfrost Plus slides. Sections are pre-incubated in binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 0.1% BSA) for 15 min. Slides are then incubated with 1-5 nM biotin-PTH(1-34) in binding buffer for 90 min at 25degC. Non-specific binding is determined in the presence of 1 uM unlabeled PTH(1-34). After incubation, sections are washed 3 × 5 min in ice-cold binding buffer, then 2 × 2 min in distilled water. Bound biotin-PTH is detected by incubating with HRP-conjugated streptavidin (1:500) for 60 min at 25degC, followed by DAB chromogen (3,3'-diaminobenzidine) for 5-10 min. Sections are dehydrated and coverslipped. Slides are visualized under brightfield microscopy. For fluorescence-based autoradiography, use fluorescent streptavidin (Alexa Fluor 555, 1:200) and mount with Fluoromount-G with DAPI. Images are captured with a fluorescence microscope or confocal. For ligand purification: cells are lysed, and streptavidin-agarose or magnetic beads are used to pull down the biotin-PTH/PTH1R complex. The eluted complex is analyzed by Western blot or mass spectrometry.
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| ADME/Pharmacokinetics |
Biotin-PTH(1-34) is not intended for in vivo PK studies. In typical receptor-binding experiments, the working concentration is 1-10 nM. The biotin-streptavidin interaction is one of the strongest non-covalent bonds (Kd ~10-¹⁴ M), ensuring stable capture. The compound is stable for up to 2 years when stored as a lyophilized powder at -20degC, protected from light and moisture. Reconstituted solutions (in sterile water or PBS with 0.1% BSA, 1-10 uM stock) are stable for up to 3 months at -20degC; avoid repeated freeze-thaw cycles.
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| Toxicity/Toxicokinetics |
No toxicity data are reported for the biotinylated probe. As a tool for in vitro receptor labeling, it is used at picomolar to low nanomolar concentrations, which are non-toxic to cultured cells. The peptide is not intended for therapeutic use. Standard laboratory safety precautions (gloves, lab coat, eye protection) are sufficient.
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| References |
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| Additional Infomation |
Biotinylated PTH(1-34) is a research-use-only product and is not approved for clinical or therapeutic applications. It is a specialized tool for studying PTH/PTH1R interactions in vitro and ex vivo. The biotin tag allows for easy detection using commercially available streptavidin conjugates (e.g., HRP, AP, fluorescent dyes, gold nanoparticles). This avoids the safety, cost, and regulatory concerns associated with radioactivity. The human sequence of PTH(1-34) (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF) is identical to teriparatide (Forteo®), an FDA-approved drug for osteoporosis; thus, the unlabeled PTH(1-34) is a known therapeutic, but the biotinylated version is strictly a research probe.
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| Molecular Formula |
C191H305N57O53S3
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| Molecular Weight |
4344.10
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| Appearance |
Solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.2302 mL | 1.1510 mL | 2.3020 mL | |
| 5 mM | 0.0460 mL | 0.2302 mL | 0.4604 mL | |
| 10 mM | 0.0230 mL | 0.1151 mL | 0.2302 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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