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Thiol detection assay kit

Manufactured by Cayman Chemical
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The Thiol Detection Assay Kit is a laboratory equipment used for the quantitative determination of thiol-containing compounds. It provides a colorimetric method to measure the concentration of thiols in a sample.

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Lab products found in correlation

Glomax discover plate reader, by Promega (1 mentions) Sonifier 450, by Emerson (1 mentions) Vibra cell vcx130, by Sonics (1 mentions) Flexstation 3, by Molecular Devices (1 mentions)

7 protocols using thiol detection assay kit

1

Quantification of Intracellular Thiols

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The intracellular thiol concentration was measured using thiol detection assay kit (Cayman Chemical Company, MI, USA) following manufacturer’s instructions. Briefly, 1x107 late log phase promastigotes were harvested by centrifugation at 2000 g at 4°C for 10 min. After washing with 1x PBS (pH 7.4) cell pellet was homogenised in 0.5 ml cold buffer (100 mM Tris-HCI, pH 7.5, containing 1mM EDTA) and centrifuged at 10,000g for 15 min at 4°C. Supernatant was collected for the fluorometric estimation of thiol at 390 nm excitation and 520 nm emission wavelengths. Thiol concentration was estimated in 100 μl of supernatant distributed into 96 well plate from three independent preparations and calculated by using the equation obtained from the linear regression of the standard curve, substituting adjusted fluorescence value for each sample as follows
Thiol concentration (nmol/107promastigotes)=Adjusted sample florescence-(y intercept)  × sample dilutionslope
Deep D.K., Singh R., Bhandari V., Verma A., Sharma V., Wajid S., Sundar S., Ramesh V., Dujardin J.C, & Salotra P. (2017). Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress. PLoS Neglected Tropical Diseases, 11(6), e0005641.
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2

H. pylori Thiol Detection Assay

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H. pylori were grown overnight in liquid culture to an OD600 of 1.0, then incubated with either 0.30 μM or 5.0 μM compounds 1–6, amoxicillin, or no inhibitor for four hours. Bacteria were washed twice with PBS, resuspended in PBS, and lysed via sonication using a Branson Sonifier 450. Thiols were detected and quantified using the Thiol Detection Assay Kit (Cayman Chemicals) and GloMax Discover Plate Reader (Promega) using an excitation wavelength of 365 nm and an emission wavelength range of 500–550 nm.
Epstein T.D., Wu B., Moulton K.D., Yan M, & Dube D.H. (2019). Sugar-modified analogs of auranofin are potent inhibitors of the gastric pathogen Helicobacter pylori. ACS infectious diseases, 5(10), 1682-1687.
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3

Quantifying Thiol Changes in P. aeruginosa

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P. aeruginosa PAO1 was obtained from a single colony and grown overnight in CAMHB to OD600 ~1.0. The culture was diluted to OD600 ~0.5 and incubated with 50 μM of Au compound for 5 h. The cultures were then pelleted (7,000 × g for 5 minutes), washed twice with PBS (1.0 mL), and then resuspended in PBS (3 mL). The bacteria were lysed via probe sonication using Vibra-Cell VCX 130 (Sonics) with a pulse of 9 s on and 9 s off at 30% amplitude for 5 min. Thiols were detected and quantified using the Thiol Detection Assay Kit (Cayman Chemicals) at an excitation wavelength of 365 nm and an emission wavelength of 520 nm.
Ndugire W., Raviranga N.G., Lao J., Ramström O, & Yan M. (2021). Gold Nanoclusters as Nano-Antibiotic Auranofin Analogues. Advanced healthcare materials, 11(9), e2101032.
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4

Thiol Content Quantification Protocol

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The free thiol contents were measured with a thiol detection assay kit (Cayman Chemical, Ann Arbor, MI) as previously described [23 (link)]. Briefly, equal amount of protein from tissue or cell lysates were mixed with 50 µL of diluted fluorometric thiol detector in 96-well plate for 5 min at dark. The fluorescence was read at an excitation wavelength of 340 nm and an emission wavelength of 520 nm using a FLUOstar OPTIMA reader. Free thiol content was expressed as µmol/mg proteins.
Barrow K., Wang Y., Yu R., Zhu J, & Yang G. (2022). H2S protects from oxidative stress-driven ACE2 expression and cardiac aging. Molecular and Cellular Biochemistry, 477(5), 1393-1403.
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5

Quantification of Liver Thiol Content

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The Thiol Detection Assay Kit (Cat no: 700340, Cayman Chemical, Ann Arbor, MI, USA) was used to determine liver free thiol content according to the manufacturer’s instructions and presented as nM per mg protein.
Chen H.J., Yip T., Lee J.K., Juliani J., Sernia C., Hill A.F., Lavidis N.A, & Spiers J.G. (2020). Restraint Stress Alters Expression of Glucocorticoid Bioavailability Mediators, Suppresses Nrf2, and Promotes Oxidative Stress in Liver Tissue. Antioxidants, 9(9), 853.
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6

Quantifying Glutathione Content

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Glutathione content was determined, in triplicate, using the Thiol Detection Assay Kit from Cayman Chemical per manufacturer’s instructions. The fluorescent signal was recorded using a FlexStation® 3 spectrophotometer (Molecular Devices). The gluathione content in each sample was estimated from the standard curve and normalized to protein content.
Yang H., Jenni S., Colovic M., Merkens H., Poleschuk C., Rodrigo I., Miao Q., Johnson B.F., Rishel M.J., Sossi V., Webster J.M., Bénard F, & Schaffer P. (2017). 18F-5-Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast Cancer Models. Journal of Nuclear Medicine, 58(3), 367-373.
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7

Thiol Depletion in S. suis Upon Antibiotic Treatment

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S. suis cells were treated with the appropriate concentrations of auranofin or ampicillin for 15 min. Subsequently, the cells were washed three times with PBS and then resuspended in 100 mM potassium phosphate (monobasic) containing 1 mM ethylenediaminetetraacetic acid (pH 7.4) and lysed using a Precellys 24 homogenizer (Bertin Corp, 2096 Gaither Rd, Rockville, MD, USA). Thiol depletion was determined using a Thiol Detection Assay kit (Cayman Chemical, Ann Arbor, MI, USA).
Lu H., Lu W., Zhu Y., Wang C., Shi L., Li X., Wu Z., Wang G., Dong W., Tan C, & Liu M. (2020). Auranofin Has Advantages over First-Line Drugs in the Treatment of Severe Streptococcus suis Infections. Antibiotics, 10(1), 26.
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