Kinetic enzyme assay kit

Manufactured by Salimetrics

Sourced in United States

The Kinetic Enzyme Assay Kit is a laboratory instrument used to measure the activity of enzymes in a sample. It provides a quantitative assessment of enzyme kinetics by tracking the rate of a specific enzymatic reaction over time.

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Lab products found in correlation

Salivette method, by Sarstedt (1 mentions) High sensitivity salivary cortisol enzyme immunoassay kit, by Salimetrics (1 mentions) Absorption reader, by Agilent Technologies (1 mentions) Orion star a111, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Salivary α-amylase kinetic enzyme assay kit α-Amylase Kinetic Enzyme Assay Kit Salivary Alpha-amylase Kinetic Enzyme Assay Kit

6 protocols using kinetic enzyme assay kit

Evaluating Salivary Biomarkers After Coffee Intake

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The caffeine content of the coffee beverages was determined using high performance liquid chromatography (HPLC) [49 (link)]. Fasting salivary samples for measurement of salivary gastrin, salivary cortisol and sAA were collected 15 min after the volunteers’ arrival. Immediately afterward, the volunteers consumed one of the four test coffee beverages, and salivary samples were collected at 15, 30, and 60 min after coffee consumption for gastrin and sAA evaluation and at 60, 120 and 180 min for cortisol evaluation.
Salivary samples were collected using the «Salivette» method (Sarstedt AG and Co, Germany). Before the collection of the salivary samples, volunteers washed their mouths well with clear water to avoid contamination of saliva samples with food components and to avoid activation of salivary flow or protein production by gustatory stimuli [44 (link)]. Then, they were asked to remove the cotton from the tube and to move it around in a circular pattern for approximately 1 min to collect saliva from all glands [44 (link)]. The tubes were stored immediately at – 20 °C.
Salivary gastrin was measured with an ELISA immunoenzymic test (Abcam ltd, UK), sAA with kinetic enzyme assay kit (Salimetrics, UK) and salivary cortisol with an ELISA immunoenzymic test (Salimetrics, UK). All biochemical measurements were performed at Agricultural University of Athens.

Papakonstantinou E., Kechribari I., Sotirakoglou Κ., Tarantilis P., Gourdomichali T., Michas G., Kravvariti V., Voumvourakis K, & Zampelas A. (2016). Acute effects of coffee consumption on self-reported gastrointestinal symptoms, blood pressure and stress indices in healthy individuals. Nutrition Journal, 15, 26.

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Salivary Biomarker Collection during Stress

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Individual saliva samples were collected from each participant 2 min before and 1-, 10-, 20-, 30- and 45-min after the TSST. Saliva was collected using Salivette® Cortisol, code blue collection tubes (Sarstedt, Nuembrecht, Germany). Briefly, participants gently moved the swab from the Salivette® in the mouth for approximately 1 min to stimulate salivation and to ensure the swab was soaked thoroughly in saliva. The swab containing the absorbed saliva was then returned to the Salivette® and the cap was replaced. All saliva samples collected during the TSST were stored frozen at −20 °C until analysis. Salivary cortisol levels were determined using a high sensitivity salivary cortisol enzyme immunoassay kit (Salimetrics, PA, USA). Salivary AA levels were determined using a kinetic enzyme assay kit (Salimetrics). All samples were analyzed at daacro.

Patterson E., Griffin S.M., Ibarra A., Ellsiepen E, & Hellhammer J. (2020). Lacticaseibacillus paracasei Lpc-37® improves psychological and physiological markers of stress and anxiety in healthy adults: a randomized, double-blind, placebo-controlled and parallel clinical trial (the Sisu study). Neurobiology of Stress, 13, 100277.

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Salivary Alpha-Amylase Activity Assay

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Saliva samples collected at both sessions were stored at −20°C until assay. Immediately prior to assay, samples were thawed to room temperature, vortexed, and centrifuged at 1,500g for 15 min. sAA activity was assayed using a commercially available kinetic enzyme assay kit (Salimetrics LLC, State College, PA) following the manufacturer’s instructions. Briefly, saliva samples were diluted to 1:200 in assay diluent and mixed with preheated substrate reagent at 37°C. The optical densities, after precisely 1 and 3 min, were measured at 405 nm using an absorption reader (BioTek Instruments Inc., Winooski, VT). sAA activity (U/mL) was calculated from the optical density change between the two readings. Intra- and inter-assay coefficients of variations were 1.7% and 4.6%, respectively.

Liu J., Kamin H.S., Kurtevski S., Kelly M, & Kertes D.A. (2019). The impact of maternal stress on infant alpha-amylase is buffered by high infant regulation and low infant negative reactivity. Developmental psychobiology, 61(8), 1204-1213.

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Quantifying Salivary α-Amylase Activity

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The salivary α-amylase activity was determined to utilize a kinetic enzyme assay kit, specifically designed and validated for the kinetic measurement of salivary α-amylase activity (Salimetrics, State College, PA, USA). This method uses a chromogenic substrate (2-chloro-p-nitrophenol linked with maltotriose) that is converted by salivary α-amylase to a product (2-chloro-p-nitrophenol) that can be spectrophotometrically measured^{54 (link)}. The absorbance of each 96-well plate was photometrically quantified using an ELISA plate reader at 405 nm wavelength (EPOCH, BioTek Instruments, Inc., Winooski, VT, USA). The increase in absorbance is directly proportional to the increase in α-amylase activity. We calculated these values based on the standardization of the kit curve and according to the manufacturer's instructions. The activity of α-amylase was expressed in unit activity per milligram of total protein (U/mg).

Lima-Holanda A.T., de Sousa E.T., Nobre-dos-Santos M, & Steiner-Oliveira C. (2021). The role of mechanical control of biofilm in the salivary pH after sucrose exposure in children with early childhood caries. Scientific Reports, 11, 7496.

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Rapid Biomarker Analysis of Salivary Samples

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EBC samples from both devices were placed on ice in a light-protected cooler (−20 °C). Within 3 min, samples were stored at −80 °C until subsequent assay, except for one microcentrifuge tube containing 1 mL of a subject’s sample used in pH testing. The pH testing (ThermoScientific Orion Star A111, Waltham, MA, USA) was conducted directly after sample collection per the manufacturer’s instructions to avoid alterations that may occur when samples are left open to room air (i.e., increases in pH due to CO₂ and other dissolved gases lost into the atmosphere).
Salivary α-amylase was assayed using a kinetic enzyme assay kit (Salimetrics, LLC, State College, PA, USA). Similarly, oxidative stress biomarkers (8-isoprostane and Myeloperoxidase) were assayed using enzyme-linked immunoabsorbent assay (ELISA) kits (Cayman, Ann Arbor, MI, USA). Finally, a pro-inflammatory biomarker (Pentraxin-3) was assayed using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). All kits were performed in accordance with the manufacturer’s instructions.

Sol J.A, & Quindry J.C. (2022). Application of a Novel Collection of Exhaled Breath Condensate to Exercise Settings. International Journal of Environmental Research and Public Health, 19(7), 3948.

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Salivary Alpha Amylase Stress Response

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The passive drool technique was implemented to collect saliva. Participants were instructed to allow saliva to pool in the bottom of their mouth, and then pass the saliva through a SalivaBio Collection Aid (SCA) into a 4ml polypropylene tube for 2 minutes. Samples were taken at baseline, prestressor, post stressor, in addition to +10min and +20min post stressor. As alpha amylase has a diurnal profile (O'Donnell, Kammerer, O'Reilly, Taylor & Glover, 2009) (link), testing occurred at 2pm each day (14:00). Samples were immediately stored at -20C and transferred to -80C within 24 hours. On day of assay, samples were completely thawed and centrifuged at 1500 × g for 15 minutes. Assays were conducted in-house by a trained technician using a kinetic enzyme assay kit provided by Salimetrics.
Saliva flow rate (mL/min) was calculated and alpha amylase output (U/min) computed for each sample. All samples were assayed in duplicate. Intra-assay variation (CV) was computed for the mean of duplicate samples and those with a CV above 15% excluded from analyses. This resulted in 56 participants providing full alpha amylase data (31 non-Type D, 25 Type D). Inter-assay variation was below 15% and therefore deemed acceptable.

Allen S.F., Wetherell M.A, & Smith M.A. (2019). An experimental investigation into cardiovascular, haemodynamic and salivary alpha amylase reactivity to acute stress in Type D individuals. Stress (Amsterdam, Netherlands), 22(4).

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