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Insulin elisa kit

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The Insulin ELISA kit is a laboratory diagnostic tool designed to quantify the concentration of insulin in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and measure insulin levels.

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Lab products found in correlation

Bca protein assay, by Thermo Fisher Scientific (4 mentions) Rpmi 1640, by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) D glucose, by Merck Group (2 mentions) Anti phospho akt ser473, by Cell Signaling Technology (2 mentions) Triglyceride assay kit, by Abcam (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Sodium pyrophosphate, by Merck Group (1 mentions) Glutamic acid, by Merck Group (1 mentions) Sepiapterin, by Merck Group (1 mentions) Pyruvate, by Merck Group (1 mentions) Malic acid, by Merck Group (1 mentions) Succinic acid, by Merck Group (1 mentions) Collagenase, by Merck Group (1 mentions) Oligomycin, by Merck Group (1 mentions) Digitonin, by Merck Group (1 mentions) Taurine, by Merck Group (1 mentions) Trypsin inhibitor, by Merck Group (1 mentions) Immobilon p pvdf membrane, by Merck Group (1 mentions)

52 protocols using insulin elisa kit

1

Insulin and Glucagon Quantification

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To determine the extent of insulin and glucagon secretion, TTFs transduced with five transcription factors were cultured in medium without phenol red. After stimulation with 15 mM glucose, 30 mM KCl and 15 mM arginine for different times, culture supernatant was collected. The amount of glucagon and insulin in the supernatant was determined by the mouse glucagon and insulin ELISA kits, respectively (Alpco Diagnostics, Salem, NH, USA), according to the manufacturer’s instructions.
Liu T., Sun L., Jiang B., Li L., Cen J., Chen X., Zhang Z., Wang Q., Cheng X., Shi Y, & Hui L. (2017). Lineage conversion of mouse fibroblasts to pancreatic α-cells. Experimental & Molecular Medicine, 49(6), e350-.
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2

Glucose-stimulated Insulin Secretion Assay

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INS-1 cells were seeded at 1 × 105 cells/well 24-well plates and treated with the indicated compounds for 24 h. Cells were then washed with Krebs buffer (1% w/v BSA in 119 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, 10 mM HEPES, pH 7.4). The cells were incubated with 500 μL Krebs buffer containing 2.5 mM glucose for 1 h and then with Krebs buffer containing 2.5 mM or 20 mM glucose for 45 min. The supernatants were collected, and secreted insulin was measured with insulin ELISA kits (ALPCO Diagnostics, Salem, NH). Cells were lysed with lysis buffer containing protease inhibitors, and total cellular protein was determined with the Bradford assay. Data are presented as the glucose-stimulated insulin secretion index: (insulin secretion stimulated by 20 mM glucose [mean of triplicate wells]/insulin secretion stimulated by 2.5 mM glucose [mean of triplicate wells]).
Duan H., Arora D., Li Y., Setiadi H., Xu D., Lim H.Y, & Wang W. (2016). Identification of 1,2,3-triazole derivatives that protect pancreatic β cells against endoplasmmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression. Bioorganic & medicinal chemistry, 24(12), 2621-2630.
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3

Glucose, Triglyceride, and Insulin Assay Protocol

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Glucose was measured using a FreeStyle Lite® (Abbott) glucometer with accompanying strips, and triglyceride assay reagents were procured from Sigma. Insulin (Humulin R, Lilly) was purchased at a local pharmacy. Insulin ELISA kits were from ALPCO Diagnostics. S-Nitro-N-acetyl-DL-penicillamine (SNAP) and cyclic guanosine monophosphate (cGMP) were procured from Cayman Chemical Company. Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, potassium phosphate, D-glucose, collagenase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, K-lactobionate, taurine, potassium phosphate, HEPES, digitonin, pyruvate, malic acid, glutamic acid, adenosine diphosphate, succinic acid, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, phenylephrine and ACh, trypsin inhibitor, and cytochrome c were procured from Sigma-Aldrich (MO, USA). Mammalian Protein Extraction Reagent (M-PER) was from Thermo Scientific, and Immobilon-P PVDF membrane was from Millipore. Sepiapterin was procured from Sigma-Aldrich.
Keller A.C., Knaub L.A., Scalzo R.L., Hull S.E., Johnston A.E., Walker L.A, & Reusch J.E. (2018). Sepiapterin Improves Vascular Reactivity and Insulin-Stimulated Glucose in Wistar Rats. Oxidative Medicine and Cellular Longevity, 2018, 7363485.
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4

GSIS Assay for Insulin Secretion

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The GSIS assay was conducted as described previously29 (link). Briefly, islets freshly isolated from mice were cultured in 24-well plates and preincubated in Krebs–Ringer bicarbonate (KRB) solution containing 3 mmol/l glucose for 30 min at 37 °C. Then, the supernatant was replaced with 400 μl of the above KRB solution and incubated for 1 h at 37 °C. Following plate centrifugation (1000 rpm, 5 min), the supernatants were collected (marked as low glucose). Next, a fresh KRB solution containing 17 mmol/l glucose was added at 400 μl per well. Following another hour of incubation at 37 °C, the supernatants were collected. Insulin concentrations in the supernatants were measured using insulin ELISA kits following the instructions (ALPCO Diagnostics, Salem, NH). For insulin measurement in islets, freshly isolated islets were homogenized in acid-ethanol solution (0.2 M HCl in ethanol) at 4 °C overnight. The supernatant was collected by centrifugation at 4000 rpm for 20 min at 4 °C. The level of insulin was measured using the same ELISA kits as above, following the manufacturer’s instructions.
Zhang K., Bao R., Huang F., Yang K., Ding Y., Lauterboeck L., Yoshida M., Long Q, & Yang Q. (2021). ATP synthase inhibitory factor subunit 1 regulates islet β-cell function via repression of mitochondrial homeostasis. Laboratory investigation; a journal of technical methods and pathology, 102(1), 69-79.
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5

Measuring Insulin Secretion in INS-1 Cells

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Static glucose stimulated insulin secretion was measured as previously described65 (link). Briefly, INS-1 cells were incubated in glucose-free RPMI-1640 culture medium for 2 h. Cells were starved for 1 h in glucose-free KRBH buffer. INS-1 cells were incubated in 2 mM glucose in KRBH for 1 h (basal samples), followed by 1 h in 20 mM glucose in KRBH (glucose-stimulated samples). Incubations were performed at 37 °C in an air incubator. Total insulin samples were taken after 1% Triton X-100 extraction. To measure the basal and stimulated insulin secretion separately, secreted insulin was normalized to total protein. Insulin samples were measured by insulin ELISA kits (Alpco Diagnostics).
Ward M.G., Li G., Barbosa-Lorenzi V.C, & Hao M. (2017). Stigmasterol prevents glucolipotoxicity induced defects in glucose-stimulated insulin secretion. Scientific Reports, 7, 9536.
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6

Akita Mouse Islet Insulin Secretion

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20 primary islets isolated from Akita mice treated with STF or vehicle were seeded in 96-well plates overnight and then incubated in fresh KRBH buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 2% w/v BSA, pH 7.4) containing 2.5 mM glucose for 1 h. Islets were incubated for an additional hour in KRBH buffer containing 2.5 or 16.7 mM glucose. Secreted insulin and proinsulin levels were measured with insulin ELISA kits (ALPCO, Salem, NH) and Rat/Mouse Proinsulin kit (Mercodia), respectively and normalized to total protein of cell lysates.
Herlea-Pana O., Eeda V., Undi R.B., Lim H.Y, & Wang W. (2021). Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes. Frontiers in Endocrinology, 12, 749879.
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7

Glucose-Stimulated Insulin Secretion Assay

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Handpicked islets were allowed to recover in RPMI without FBS overnight, as described previously [23 ]. The islets were washed with Krebs solution containing the following (mM): 2.8 glucose, 102 NaCl, 5 KCl, 1.2 MgCl2, 2.7 CaCl2, 20 HEPES, 5 NaHCO3, and 10 mg/mL BSA (pH 7.4). They were incubated at 37°C for 45 min and roughly 10 islet equivalents were transferred into the wells of a 12-well plate containing 1 mL pre-warmed KRB solution. GSIS was measured by calculating the total percentage of insulin secreted within a 45-minute treatment duration. Technical replicates were conducted for each experiment. Insulin ELISA kits (ALPCO, Salem, NH) were used to measure the insulin secretion, as per the manufacturer's instructions.
Bosma K.J., Andrei S.R., Katz L.S., Smith A.A., Dunn J.C., Ricciardi V.F., Ramirez M.A., Baumel-Alterzon S., Pace W.A., Carroll D.T., Overway E.M., Wolf E.M., Kimple M.E., Sheng Q., Scott D.K., Breyer R.M, & Gannon M. (2021). Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances β-cell proliferation and identity and relieves oxidative damage. Molecular Metabolism, 54, 101347.
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8

GSIS Assay for Insulin Secretion

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The GSIS assay was conducted as described previously29 (link). Briefly, islets freshly isolated from mice were cultured in 24-well plates and preincubated in Krebs–Ringer bicarbonate (KRB) solution containing 3 mmol/l glucose for 30 min at 37 °C. Then, the supernatant was replaced with 400 μl of the above KRB solution and incubated for 1 h at 37 °C. Following plate centrifugation (1000 rpm, 5 min), the supernatants were collected (marked as low glucose). Next, a fresh KRB solution containing 17 mmol/l glucose was added at 400 μl per well. Following another hour of incubation at 37 °C, the supernatants were collected. Insulin concentrations in the supernatants were measured using insulin ELISA kits following the instructions (ALPCO Diagnostics, Salem, NH). For insulin measurement in islets, freshly isolated islets were homogenized in acid-ethanol solution (0.2 M HCl in ethanol) at 4 °C overnight. The supernatant was collected by centrifugation at 4000 rpm for 20 min at 4 °C. The level of insulin was measured using the same ELISA kits as above, following the manufacturer’s instructions.
Zhang K., Bao R., Huang F., Yang K., Ding Y., Lauterboeck L., Yoshida M., Long Q, & Yang Q. (2021). ATP synthase inhibitory factor subunit 1 regulates islet β-cell function via repression of mitochondrial homeostasis. Laboratory investigation; a journal of technical methods and pathology, 102(1), 69-79.
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9

Plasma Insulin Measurement from Blood

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Blood was collected by cardiac puncture in EDTA-coated tubes. Samples were then centrifuged at 2,000g for 15 min to remove red blood cells and plasma insulin was measured using Insulin ELISA KIT (Alpco, 80-INSMS-E01) per the manufacturer’s instructions.
Zani F., Blagih J., Gruber T., Buck M.D., Jones N., Hennequart M., Newell C.L., Pilley S.E., Soro-Barrio P., Kelly G., Legrave N.M., Cheung E.C., Gilmore I.S., Gould A.P., Garcia-Caceres C, & Vousden K.H. (2023). The dietary sweetener sucralose is a negative modulator of T cell-mediated responses. Nature, 615(7953), 705-711.
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10

Insulin Delivery Efficiency in Diabetic Mice

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Diabetic mice were divided into the following 4 groups: (a) an untreated group (negative control); (b) a subcutaneous injection group (0.2 IU, positive control); (c) a patch group (DMNs loaded with 0.2 IU insulin); and (d) a Microlancer group (DMNs loaded with 0.2 IU insulin) (n = 5 per group). Mice were fasted throughout the experiment, but were allowed free access to water. After the back hair was removed from each mouse using an electric shaver, 0.2 IU insulin was administered using the DMN patch, the Microlancer or a SC injection. Blood samples (0.1 ml) were collected hourly from tail-vein lacerations for 6 h post-application. Plasma was then separated by centrifuging the samples at 10,000 rpm for 15 min. The serum samples were immediately frozen at −80°C until analysis. The plasma glucose levels over time for each group were measured using a Glucose CII-Test kit. The plasma insulin concentrations were determined using an insulin ELISA kit (ALPCO).
Lahiji S.F., Dangol M, & Jung H. (2015). A patchless dissolving microneedle delivery system enabling rapid and efficient transdermal drug delivery. Scientific Reports, 5, 7914.
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