Aprotinin

Manufactured by Phoenix Pharmaceuticals

Sourced in United States

Aprotinin is a protease inhibitor used in laboratory settings. It functions by inhibiting a range of serine proteases, including trypsin, plasmin, and kallikrein.

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Lab products found in correlation

Blood collection tube, by BD (3 mentions) Quantared enhanced chemifluorescent hrp substrate, by Thermo Fisher Scientific (3 mentions) Cytofluor4000 plate reader, by Thermo Fisher Scientific (3 mentions) Dpp4 inhibitor, by Merck Group (2 mentions) Corticosterone elisa kit, by Abcam (2 mentions) Quantared enhanced chemifluorescenthrp substrate, by Abcam (2 mentions) Vacutainer tube, by BD (2 mentions) Epoch system, by Agilent Technologies (1 mentions) Dpp4 010, by Merck Group (1 mentions) Edta coated microcentrifuge tubes, by Sarstedt (1 mentions) Total glp 1, by Crystal Chem (1 mentions) Ultra sensitive elisa kit, by Crystal Chem (1 mentions) Ek 065 07, by Phoenix Pharmaceuticals (1 mentions) Vapor pressure osmometer, by Wescor (1 mentions) Dipeptidyl peptidase 4 inhibitor, by Merck Group (1 mentions) Murine lps elisa kit, by Cusabio (1 mentions) Inactin, by Merck Group (1 mentions) Ria kit, by Linco Research (1 mentions) Eia kit, by Phoenix Pharmaceuticals (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)

12 protocols using aprotinin

Ghrelin Measurement Protocol Using ELISA

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Blood samples were collected into EDTA-containing tubes and then aprotinin (Phoenix Pharmaceuticals, California, USA) was added immediately. The blood was centrifuged at 1600 x g for 15 minutes; after separation of the plasma, it was stored at −80°C until the ghrelin assessment. Measurements of ghrelin were performed in an EPOCH system (BioTek Instruments, Inc, USA) using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals, California, USA) following the manufacturers’ instructions. The test range of the ghrelin ELISA kit was 0-100 ng/mL. The specimens were run together in the same experiment.

Ucan B., Sahin M., Kizilgul M., Ozbek M., Ozdemir S., Calıskan M, & Cakal E. (2017). Serum ghrelin levels in papillary thyroid carcinoma. Archives of Endocrinology and Metabolism, 61(5), 464-469.

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Blood Sample Collection and Hormone Analysis

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Samples were drawn into 4 ml EDTA vacutainers prepared with aprotinin (Phoenix Pharmaceuticals, Inc. Catalog #: RK-APRO): 1 mg/mL (500 KIU/mL) of blood and DPP4 inhibitor (Millipore, Catalog #: DPP4-010): 10 uL/mL of blood. After collection, samples were mixed by gentle inversion of the tube. The tube was then placed in an ice bath and centrifuged within 1 h of collection at 1500–2000 × g (RCF), for 15 min at 4 °C. Plasma (0.5 mls) was aliquoted into each of four cryovials. Cryovials were then immediately frozen and stored upright at −70 °C until assay. Batched assay was performed using a Millipore Human Metabolic Expanded Hormone Magnetic Bead Panel (Cat#HMHEMAG-34K-04, Insulin, C-peptide, GLP-1 (total). Plasma glucose concentrations were determined using an YSI 2300 autoanalyzer (Yellow Springs Instruments, Yellow Springs, OH).

Beale E., Lim E., Yassine H., Azen C, & Christopher C. (2019). Development of a delayed-release nutrient for appetite control in adults with obesity and type 2 diabetes and initial clinical testing in a single dose randomized controlled trial. Nutrition & Diabetes, 9, 20.

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Plasma ACTH and Corticosterone Quantification

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Following sacrifice of mice by cervical dislocation and decapitation,
trunk blood was collected directly into blood collection tubes (Becton
Dickinson) containing 50 μl aprotinin (Phoenix Pharmaceuticals). Plasma
was obtained by centrifugation at 1600 × g for 15 minutes at
4°C, and stored at −80°C. Plasma ACTH concentrations
were measured using the ACTH ELISA kit (MD Biosciences), according to the
manufacturer’s instructions, with the following modifications: 1) 100
μl of the controls or blood plasma combined with 100 μl of
phosphate buffered saline (PBS) (pH 7.4) were used in place of 200 μl
plasma and 2) the results were assessed with the QuantaRed Enhanced
Chemifluorescent HRP Substrate (Thermo Fisher). Plasma corticosterone
concentrations were measured using the corticosterone ELISA kit (Abcam),
according to the manufacturer’s instructions, with the following
modifications: 1) Plasma was diluted 25 times instead of 100 times with buffer M
and 2) the results were assessed with the QuantaRed Enhanced Chemifluorescent
HRP Substrate. Fluorescence was measured with a CytoFluor4000 plate reader
(Applied Biosystems).

Kondoh K., Lu Z., Ye X., Olson D.P., Lowell B.B, & Buck L.B. (2016). A specific area of olfactory cortex involved in stress hormone responses to predator odors. Nature, 532(7597), 103-106.

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Quantifying Extracellular Nociceptin Levels

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Extracellular nociceptin protein levels in culture supernatants were measured by fluorescent enzyme immunoassay (FEIA). After culturing with or without PMA 10 ng/ml for 0, 24, and 48 h, blood samples were collected in the presence of the protease inhibitor aprotinin 600 KIU/ml (Phoenix Pharmaceuticals, Karlsruhe, Germany) and centrifuged (16,000 g, 15 min at 4°C). Supernatants were thoroughly mixed with an equal amount of buffer A (Phoenix Pharmaceuticals, Karlsruhe, Germany) and centrifuged at 12,000 g for 20 min. C-18 SEP cartridges were used to purify small peptides from the supernatant solution, and nociceptin protein levels were determined using the Nociceptin/Orphanin FQ Fluorescent EIA kit (Phoenix Pharmaceuticals, Karlsruhe, Germany) following the manufacturer’s instructions.^{17 (link)}

Zhang L., Stüber F., Lippuner C., Schiff M, & M Stamer U. (2019). ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes. Molecular Pain, 15, 1744806919828921.

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Plasma ACTH Assay in Mice

Cited 1 time

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Plasma ACTH assays were performed as described previously^{11 (link)}. Briefly, after mice were killed by cervical dislocation and decapitation, trunk blood was collected directly into blood collection tubes (Becton Dickinson) containing 50 μL aprotinin (Phoenix Pharmaceuticals). Plasma was obtained by centrifugation at 1600×g for 15 min at 4 °C, and stored at − 80 °C. Plasma ACTH concentrations were measured using the ACTH ELISA kit (MD Biosciences), according to the manufacturer’s instructions, with the following modifications: (1) 100 μL of the controls or blood plasma combined with 100 μL of PBS (Phosphate Buffered Saline, pH 7.4) was used in the place of 200 μL plasma and (2) the results were assessed with the QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher). Fluorescence was measured with a CytoFluor4000 plate reader (Applied Biosystems). ACTH assays were carried out in duplicate. The average inter- and intra-assay coefficients of variation were 4.0% and 5.8%, respectively.

Lee E.J., Saraiva L.R., Hanchate N.K., Ye X., Asher G., Ho J, & Buck L.B. (2022). Odor blocking of stress hormone responses. Scientific Reports, 12, 8773.

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Measuring GLP-1, GLP-2, and Corticosterone in Mice

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For GLP-1 measurements, mice were fasted for 5 h and blood samples were collected from tail veins at 0, 5, 15, and 30 min following an IG.GTT (1 g/kg). For GLP-2 measurements, mice were fasted for 5 h and hepatic portal vein (HPV) samples were collected 15 min following an IG.GTT (1 g/kg) or PBS (vehicle).
The collected blood was immediately transferred to EDTA-coated microcentrifuge tubes (Sarstedt AG & Co.) containing 0.6TIU aprotinin (Phoenix Pharmaceuticals Inc.), and 10 μl of DPPIV inhibitor (EMD Millipore) per 1 mL of blood. Plasma was obtained by centrifugation at 1500 × g for 15 min at 4 °C and stored at −80 °C until measurement of total GLP-1 (Crystal Chem, Elk Grove Village, IL, USA, cat # 81508), or total GLP-2 (MyBioSource, San Diego, CA, USA). Plasma insulin was assessed with an ultra-sensitive ELISA kit (Crystal Chem, Inc, Downer's Grove, IL, USA). Plasma corticosterone was measured in mice that were fed ad libitum (0 h), or fasted for 5 h, or 16 h. Blood was collected via tail vein (25 μL) in heparin coated tubes and plasma was isolated by centrifugation at 1500 × g at 4 °C for 15 min and stored at −80 °C until the measurement of corticosterone levels (Enzo Life Sciences, Farmingdale, NY).

Smith K., Karimian Azari E., LaMoia T.E., Hussain T., Vargova V., Karolyi K., Veldhuis P.P., Arnoletti J.P., de la Fuente S.G., Pratley R.E., Osborne T.F, & Kyriazis G.A. (2018). T1R2 receptor-mediated glucose sensing in the upper intestine potentiates glucose absorption through activation of local regulatory pathways. Molecular Metabolism, 17, 98-111.

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Plasma Osmolality and Vasopressin Measurement

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Trunk blood (1–2 mL) was collected from each rat after decapitation and prepared for measuring plasma osmolality and haematocrit as described previously.^{18 (link)} Plasma osmolality was measured on a vapor pressure osmometer (Wescor, Logan, UT, USA). Two heparinised capillary tubes (Fisher Scientific, Hampton, NH, USA) were filled for measuring haematocrit using a micro-haematocrit capillary tube reader (Lancer, St Louis, MO, USA).
Further blood (5–6 mL) was collected in Vacutainer tubes (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) containing the anticoagulant ethylenediaminetetraacetic acid (12 mg). The proteinase inhibitor, aprotinin (0.6 TIU mL⁻¹ of blood; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) was added and the sample was centrifuged at 1600 g for 15 minutes at 4°C. A sample of plasma (2–3 mL) was removed from each sample and peptides were extracted from plasma by solid phase extraction using a C-18 SEP-Column (Phenomenex, Torrance, CA, USA). After extraction, each sample was subjected to vacuum centrifugal concentration. Circulating AVP concentration was measured using a specific enzyme-linked immunosorbent assay in accordance with the manufacturer’s instructions (EK-065–07; Phoenix Pharmaceuticals, Inc.). Four parametric logistic analysis was performed to quantify the concentration of peptide.

Balapattabi K., Little J.T., Farmer G.E, & Cunningham J.T. (2018). High salt loading increases brain derived neurotrophic factor in supraoptic vasopressin neurones. Journal of neuroendocrinology, 30(11), e12639.

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Plasma GLP-1, GIP, and LPS Quantification

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Following a 4-h fast, mice were administered an oral dextrose bolus and anesthetized via 5% isopropanol inhalation several minutes before euthanization. Mice were then euthanized by cardiac puncture 15 min after administration of oral glucose bolus and whole blood was collected and stored in heparinized blood collection tubes. A cocktail of protease inhibitors including dipeptidyl peptidase-4 inhibitor (Sigma-Aldrich) and aprotinin (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) was immediately added, and samples were stored on ice for 30 minutes before centrifugation (600 g) at 4°C for 15 min. Plasma portions were isolated for ELISA assays. Plasma GLP-1 levels and GIP levels were quantified by Mouse Total GLP-1 ELISA kit (CrystalChem, Elk Grove Village, IL, USA) and Mouse Total GIP ELISA kit (Millipore), respectively. Lipopolysaccharide (LPS) levels were measured with the murine LPS ELISA kit (Cusabio, Wuhan, China).

Lednovich K.R., Gough S., Priyadarshini M., Pandya N., Nnyamah C., Xu K., Wicksteed B., Mishra S., Jain S., Zapater J.L., Cordoba-Chacon J., Yadav H, & Layden B.T. (2024). Intestinal FFA2 promotes obesity by altering food intake in Western diet-fed mice. The Journal of Endocrinology, 260(2), e230184.

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Rat Plasma AVP and Osmolality Measurement

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After the final treatment week for each drinking study, rats were administered intraperitoneally CNO and subcutaneously ANG II or 3% HTN, and denied food and water access the following 90 min. Animals were then anesthetized using 100-mg/kg inactin (Sigma-Aldrich) intraperitoneally. Blood was collected by cardiac puncture (3 ml) and transferred to an EDTA vacutainer containing 100 µl of aprotinin (catalog #RK-APRO, Phoenix Pharmaceuticals, Inc.) per milliliter of blood (300 µl total) immediately preceding the perfusion to measure plasma AVP and osmolality. Rats were transcardially flushed first with PBS and then perfused using 4% PFA. Brain tissue was fixed overnight in 4% PFA before being dehydrated in 30% sucrose.

Marciante A.B., Wang L.A., Farmer G.E, & Cunningham J.T. (2019). Selectively Inhibiting the Median Preoptic Nucleus Attenuates Angiotensin II and Hyperosmotic-Induced Drinking Behavior and Vasopressin Release in Adult Male Rats. eNeuro, 6(2), ENEURO.0473-18.2019.

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Measuring Plasma Hormones: Ghrelin, Obestatin, Insulin

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For active ghrelin and des-acyl ghrelin (DAG): about 200 μl blood was collected in EDTA-coated capillary tubes with or without 18 hours fasting. After spin down, plasma was transferred to an ice-cold tube with 5 μl 1N HCl. Then, 1 μl fresh PMSF (10 mg/ml in methanol, Sigma, St. Louis, MO) was added and gently mixed. 25 μl plasma was measured with either RIA assay kits (EMD Millipore, Billerica, MA) or ELISA kits (Mitsubishi Kagaku Iatron, Tokyo, Japan). For obestatin: about 200 μl blood was collected in EDTA-coated capillary tubes. The blood sample was then transferred to tubes and mixed with 20 μl aprotinin (6 TIU/ml, Phoenix, Milpitas, CA). After spin down, plasma was transferred to a new ice-chilled tube. 50 μl of plasma was measured with an EIA kit (Phoenix, Milpitas, CA). For insulin: about 200 μl blood was collected in EDTA-coated capillary tubes. The blood sample was then transferred to tubes. After spin down, plasma was transferred to a new ice-chilled tube. 50 μl of plasma was measured with a RIA kit (Linco Research, St. Charles, MI), following manufacture's instruction.

Lin L., Lee J.H., Bongmba O.Y., Ma X., Zhu X., Sheikh-Hamad D, & Sun Y. (2014). The suppression of ghrelin signaling mitigates age-associated thermogenic impairment. Aging (Albany NY), 6(12), 1019-1032.

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