GLP-1 receptor (GLP-1R). GLP-1 (7-36)-Lys(Biotin), amide, human is a biotinylated analog of the active, naturally occurring form of human GLP-1 (7-36) amide. GLP-1 is an incretin hormone secreted by intestinal L-cells in response to nutrient ingestion. It binds to and activates the GLP-1 receptor (GLP-1R), a class B G protein-coupled receptor (GPCR) expressed on pancreatic beta-cells, as well as in the brain, heart, kidney, stomach, and other tissues. Activation of GLP-1R leads to Gs protein-mediated increase in intracellular cAMP, which potentiates glucose-stimulated insulin secretion (GSIS), inhibits glucagon secretion, promotes beta-cell proliferation and survival, slows gastric emptying, and induces satiety. The biotin moiety (attached to Lysine at the C-terminus) does not interfere with GLP-1R binding or activation, as the C-terminal region is not critical for receptor recognition (the N-terminus is crucial). The amide at the C-terminus (position 36) is the native form, which is more stable and bioactive than the free acid. This biotinylated peptide is not intended as a therapeutic; it is a research reagent for studying GLP-1R pharmacology.

In vitro, Glucagon-Like Peptide 1 (GLP-1) (7-36)-Lys (Biotin), amide, human retains full biological activity comparable to native GLP-1 (7-36) amide. In GLP-1R-expressing cells (e.g., HEK293-GLP-1R, INS-1, MIN6), the biotinylated peptide induces cAMP accumulation with an EC50 in the low nanomolar range (0.1-1 nM), similar to non-biotinylated GLP-1. It also stimulates glucose-dependent insulin secretion (GSIS) from beta-cell lines and isolated islets. The biotin tag does not alter the peptide's affinity for the GLP-1R. The peptide can be used for receptor binding studies using streptavidin-based detection. For example, in a cell-based ELISA or fluorescence microscopy, the biotinylated GLP-1 binds to GLP-1 receptors on live cells, and after washing, it can be detected using fluorescently labeled streptavidin (e.g., FITC-streptavidin, Cy3-streptavidin) or HRP-streptavidin. This allows for visualization of receptor distribution and internalization. In pull-down assays, the biotinylated peptide can be immobilized on streptavidin-agarose beads to capture GLP-1R from cell lysates, allowing identification of interacting proteins. The peptide is not toxic to cells at concentrations up to 100 nM. The TFA salt is not mentioned; the compound appears to be supplied as an acetate or trifluoroacetate but is not critical. The sequence is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[K(Biotin)]-NH2.

Not applicable (research reagent). The biotinylated GLP-1 (7-36) amide is used as an in vitro research tool and is not intended for in vivo administration as a drug. It is not used in animal models for efficacy studies because the biotin tag could alter pharmacokinetics and immunogenicity. However, the peptide could theoretically be used for ex vivo imaging or for targeting GLP-1R-expressing cells in vivo if conjugated to a carrier, but this is not standard. No in vivo activity data are available for this specific biotinylated analog. The parent native GLP-1 (7-36) amide has potent glucose-lowering effects in animals (e.g., reduction of blood glucose in diabetic mice). But the biotinylated version is not used for this purpose due to cost and lack of necessity; native GLP-1 or its long-acting analogs (e.g., exenatide, liraglutide) are used. Therefore, no in vivo activity data are provided. The product is strictly a research reagent.

For non-cellular binding assays (e.g., direct binding of GLP-1 to GLP-1R), the biotinylated peptide can be used in an ELISA-based binding assay. 96-well plates are coated with streptavidin (5-10 ug/mL in PBS) overnight at 4degC. After blocking with 1% BSA, the biotinylated GLP-1 peptide (0.1-100 nM) is added and captured by streptavidin. Then, recombinant GLP-1 receptor protein (His-tagged or Fc-tagged) is added and allowed to bind. After washing, bound receptor is detected with an anti-GLP-1R antibody (or anti-tag antibody) followed by HRP-conjugated secondary antibody and TMB substrate. The absorbance at 450 nm is measured. Alternatively, the receptor can be immobilized directly, and the biotinylated peptide is added, then detected with streptavidin-HRP. For surface plasmon resonance (SPR): Biotinylated GLP-1 peptide is immobilized on a streptavidin-coated sensor chip (SA chip). Increasing concentrations of recombinant GLP-1R protein are flowed over the chip. The association and dissociation phases are recorded, and the KD is calculated. This method allows measurement of binding kinetics (kon, koff). This is a direct binding assay without cells. For a competition binding assay, a fixed concentration of biotinylated GLP-1 is incubated with varying concentrations of unlabeled GLP-1 or other agonists in the presence of GLP-1R protein, and the amount of bound biotinylated peptide is measured. The IC50 and Ki for unlabeled compounds are determined. These assays are common in drug discovery for screening GLP-1R ligands. The TFA/acetate salt does not affect these assays. The peptide is stable in solution at -20degC in PBS or water. Avoid repeated freeze-thaw.

For cell-based assays, the biotinylated GLP-1 peptide is used to visualize GLP-1R binding and internalization. HEK293 cells stably expressing human GLP-1R are seeded in 8-well chamber slides or 96-well plates (2-4 × 10^4 cells/well). After 24 h, cells are serum-starved for 2-4 h in DMEM with 0.1% BSA. Cells are then incubated with biotinylated GLP-1 peptide (1-100 nM) in binding buffer (DMEM, 0.1% BSA, 20 mM HEPES) for 30-60 min at 4degC (to prevent internalization) or at 37degC (to allow internalization). After incubation, cells are washed with cold PBS. For detection, cells are fixed with 4% paraformaldehyde for 10-15 min, permeabilized with 0.1% Triton X-100 (if internalization is to be studied), then blocked with 5% BSA. Cells are incubated with fluorescently labeled streptavidin (e.g., Alexa Fluor 488-streptavidin, 1:500) for 30-60 min at room temperature. Nuclei are counterstained with DAPI. Cells are imaged by confocal or fluorescence microscopy. At 4degC, staining is predominantly on the plasma membrane; at 37degC, punctate intracellular staining (endosomes) is observed, indicating receptor internalization. For flow cytometry: Cells are detached with non-enzymatic cell dissociation buffer, incubated with biotinylated GLP-1 (10-100 nM) for 30-60 min at 4degC, washed, then stained with streptavidin-PE or streptavidin-FITC for 30 min. After washing, cells are analyzed on a flow cytometer. This allows quantification of receptor binding. For cAMP accumulation assays, the biotinylated peptide is not necessary; unlabeled GLP-1 is used. However, the biotinylated peptide can be used to confirm that the biotin tag does not interfere with signaling: perform a cAMP HTRF assay using the biotinylated peptide (0.001-1000 nM) in GLP-1R cells and compare EC50 to unlabeled GLP-1. The EC50 should be similar (nM range). For pulldown assays to identify GLP-1R-interacting proteins: Cell lysates (from GLP-1R-expressing cells, 1-2 mg protein) are incubated with biotinylated GLP-1 peptide (0.1-1 uM) for 2-4 hours at 4degC. Then, streptavidin-agarose beads (50 uL slurry) are added and incubated overnight at 4degC. Beads are washed 3-5 times with lysis buffer, and bound proteins are eluted with SDS sample buffer, separated by SDS-PAGE, and identified by mass spectrometry or immunoblotting (e.g., with anti-GLP-1R antibody). This method can also be used to study receptor oligomerization or interactions with beta-arrestin, GRKs, or other signaling molecules. The biotinylated peptide can also be used in a competitive binding assay on live cells: cells are incubated with biotinylated GLP-1 (10 nM) plus varying concentrations of unlabeled GLP-1 or other compounds for 60 min at 4degC, then stained with streptavidin-PE, and analyzed by flow cytometry. The IC50 of the unlabeled compound is determined. All experiments should include a negative control (no peptide, no cells, or pre-incubation with excess unlabeled GLP-1 to block binding). The peptide is dissolved in water or PBS (1-10 mM stock) and stored at -20degC. Protect from light (biotin is stable, but avoid photobleaching of fluorophores if used downstream).

Not applicable (in vitro reagent). The biotinylated GLP-1 peptide is not administered to animals for efficacy studies. However, it may be used in ex vivo tissue binding assays. For example, after a mouse is euthanized, brain or pancreas sections are prepared. The sections are incubated with biotinylated GLP-1 (10-100 nM) in binding buffer for 1-2 hours at room temperature, then washed, fixed, and detected with streptavidin-HRP or fluorescent streptavidin. This allows visualization of GLP-1R distribution in tissues. No in vivo animal protocol is applicable because the compound is not dosed to live animals. If researchers want to administer the peptide intravenously to study GLP-1R targeting in vivo, they would likely use a radiolabeled ligand (e.g., 68Ga-labeled GLP-1), not a biotinylated peptide, due to the presence of endogenous biotin and avidin in serum. Therefore, no in vivo protocol is provided. The product is strictly a research reagent for in vitro use.

Not applicable (research reagent). The biotinylated GLP-1 (7-36) amide is not intended for in vivo use; therefore, no pharmacokinetic data are available. As a peptide (MW ~3.7 kDa), if administered intravenously, it would have a very short plasma half-life (minutes) due to rapid degradation by dipeptidyl peptidase-4 (DPP-4) at the N-terminus (Ala2-Glu3 cleavage) and by other proteases. The biotin tag would likely not alter this property. The peptide is not orally bioavailable. It is not used for systemic therapy, and PK studies are not applicable. The compound is stable in cell culture media for several hours at 37degC (depending on the presence of proteases), but for long-term storage, it is kept at -20degC. The peptide is typically supplied as a lyophilized powder (TFA or acetate salt). It should be reconstituted in water or PBS containing 0.1% BSA to prevent adsorption to plastic. The biotinylated peptide is used only for in vitro applications.

No toxicity data are available for the biotinylated GLP-1 (7-36) amide. The non-biotinylated GLP-1 (7-36) amide is an endogenous peptide hormone and is generally non-toxic at physiological concentrations. The biotin moiety is a vitamin (vitamin B7) and is also non-toxic. In cell-based assays, the biotinylated peptide is used at concentrations up to 100 nM-1 uM for short periods (1-24 hours) and does not cause cytotoxicity (as assessed by LDH or MTT). No genotoxicity, carcinogenicity, or reproductive toxicity studies have been performed. The peptide is for research use only and is not intended for human or veterinary use. Standard laboratory safety precautions (gloves, lab coat) are sufficient. The TFA/acetate salt is not toxic. The compound is not an approved drug.

[1]. Farnesoid X Receptor (FXR) Interacts with Camp Response Element Binding Protein (CREB) to Modulate Glucagon-Like Peptide-1 (7-36) Amide (GLP-1) Secretion by Intestinal L Cell. Cell Physiol Biochem. 2018; 47(4): 1442-1452.

GLP-1 (7-36) amide is the major active form of the incretin hormone GLP-1, accounting for approximately 80% of endogenous GLP-1. It is secreted from intestinal L-cells and acts through the GLP-1 receptor to regulate glucose homeostasis. GLP-1 receptor agonists (e.g., exenatide, liraglutide, dulaglutide, semaglutide) are approved for the treatment of type 2 diabetes and obesity. Native GLP-1 has a very short half-life due to DPP-4 degradation; DPP-4 inhibitors (e.g., sitagliptin, saxagliptin) are also approved. Biotinylation of GLP-1 is used as a non-radioactive detection method. Biotin-streptavidin interaction is one of the strongest non-covalent bonds in nature (Kd ~10-15 M). The biotin tag is usually added at the C-terminus (e.g., via a lysine residue) to avoid interference with N-terminal binding to GLP-1R, as the N-terminus is crucial for receptor activation. The C-terminal amide is also important for activity. This product is widely used in GLP-1R binding assays, internalization studies, and receptor interaction studies. It is for research use only and is not a drug. It is not approved for clinical use.

C165H252N44O48S

3649.84

Solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~4 mg/mL (~1.10 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)