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Universal sulfotransferase activity kit

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The Universal Sulfotransferase Activity Kit is a laboratory tool designed to measure the enzymatic activity of sulfotransferase, an important class of enzymes involved in various metabolic processes. The kit provides a standardized and quantitative method to assess sulfotransferase activity in biological samples.

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Lab products found in correlation

Obatoclax, by R&D Systems (2 mentions) Nadph glo assay, by Promega (2 mentions) Recombinant human sult1a1, by R&D Systems (2 mentions) Recombinant human blvrb, by R&D Systems (2 mentions) Spectramax m5, by Molecular Devices (2 mentions) Softmax pro software, by Molecular Devices (2 mentions) Blyscan, by Biocolor (1 mentions)

7 protocols using universal sulfotransferase activity kit

1

SULT1A1 and BLVRB Activity Assays

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Recombinant human SULT1A1 (R&D Systems) activity in the presence of 0.2 mM 3′-phosphoadenosine-5′-phosphosulfate and the indicated concentrations of RITA was measured by a phosphatase-coupled assay using the Universal Sulfotransferase Activity Kit (R&D Systems) according to the manufacturer’s instructions. The assay was carried out in Corning clear 96-well plates and incubated (37°C, 1 hour) before addition of detection reagents and visualization at 620 nm using a SpectraMax M5 microplate reader using the SoftMax Pro software (Molecular Devices).
Recombinant human BLVRB was purchased from R&D Systems, Inc. BLVRB activity was measured in the presence of 20 ng/μL BLVRB, obatoclax, and 0.01 mM NADPH in 100 mM sodium phosphate (pH 7.5) in a 20 μL volume in white, opaque 384-well plates. The reaction was incubated for 3 hours at 37°C before detection using the NADPH-Glo assay (Promega) according to the manufacturer’s instructions. NADPH and sodium phosphate were purchased from Sigma.
Rees M.G., Seashore-Ludlow B., Cheah J.H., Adams D.J., Price E.V., Gill S., Javaid S., Coletti M.E., Jones V.L., Bodycombe N.E., Soule C.K., Alexander B., Li A., Montgomery P., Kotz J.D., Hon C.S., Munoz B., Liefeld T., Dančík V., Haber D.A., Clish C.B., Bittker J.A., Palmer M., Wagner B.K., Clemons P.A., Shamji A.F, & Schreiber S.L. (2015). Correlating chemical sensitivity and basal gene expression reveals mechanism of action. Nature chemical biology, 12(2), 109-116.
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2

SULT1A1 and BLVRB Activity Assays

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Recombinant human SULT1A1 (R&D Systems) activity in the presence of 0.2 mM 3′-phosphoadenosine-5′-phosphosulfate and the indicated concentrations of RITA was measured by a phosphatase-coupled assay using the Universal Sulfotransferase Activity Kit (R&D Systems) according to the manufacturer’s instructions. The assay was carried out in Corning clear 96-well plates and incubated (37°C, 1 hour) before addition of detection reagents and visualization at 620 nm using a SpectraMax M5 microplate reader using the SoftMax Pro software (Molecular Devices).
Recombinant human BLVRB was purchased from R&D Systems, Inc. BLVRB activity was measured in the presence of 20 ng/μL BLVRB, obatoclax, and 0.01 mM NADPH in 100 mM sodium phosphate (pH 7.5) in a 20 μL volume in white, opaque 384-well plates. The reaction was incubated for 3 hours at 37°C before detection using the NADPH-Glo assay (Promega) according to the manufacturer’s instructions. NADPH and sodium phosphate were purchased from Sigma.
Rees M.G., Seashore-Ludlow B., Cheah J.H., Adams D.J., Price E.V., Gill S., Javaid S., Coletti M.E., Jones V.L., Bodycombe N.E., Soule C.K., Alexander B., Li A., Montgomery P., Kotz J.D., Hon C.S., Munoz B., Liefeld T., Dančík V., Haber D.A., Clish C.B., Bittker J.A., Palmer M., Wagner B.K., Clemons P.A., Shamji A.F, & Schreiber S.L. (2015). Correlating chemical sensitivity and basal gene expression reveals mechanism of action. Nature chemical biology, 12(2), 109-116.
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3

Sulfotransferase Activity Assay by TLC

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Thin-layer chromatography (TLC) was performed in the presence of PAP[35S] as the sulfate donor in the presence of several substrates [12 (link)]. The reaction mixture containing each substrate (25 μM), swSULT ST3 (1.0–2.0 μg/20 μL), and HEPES buffer (pH 7.0) was incubated at 37°C for 10 min. The reaction was stopped by heating. Then, 1 μL of the reaction mixture was applied to a cellulose or silica gel TLC plate using n-butanol/ isopropanol/formic acid/ water (3:1:1:1 by volume) as the solvent system to separate the sulfated products. We detected the spot positions corresponding to the sulfated products on the TLC using a Fluoro Image Analyzer, Typhoon FLA 9500 (Cytiva, Tokyo, Japan). The specific activity of swSULT ST3 towards various substrates was measured using a phosphatase-coupled assay with a Universal Sulfotransferase Activity Kit (R&D Systems, Minneapolis, MN, USA) in the presence of non-labelled PAPS. The assay was composed of two steps: (1) conversion of PAPS to PAP by swSULT ST3, and (2) release of inorganic phosphate by a coupling phosphatase. After incubation at 37°C for 1 h, the change in absorbance was detected at 620 nm.
Yamamoto K., Yamada N., Endo S., Kurogi K., Sakakibara Y, & Suiko M. (2022). Novel silkworm (Bombyx mori) sulfotransferase swSULT ST3 is involved in metabolism of polyphenols from mulberry leaves. PLoS ONE, 17(8), e0270804.
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4

Sulfotransferase Activity Quantification

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Sulfotransferase activity was determined using the Universal Sulfotransferase Activity Kit (R&D, Minneapolis, MN), which assesses the sulfotransferase activity of all the sulfotransferases in the cells or tissue tested. 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is the sulfate donor for the sulfotransferase reaction. PAPS (10 μl, 1 mM), acceptor substrate (10 μl), and coupling phosphatase (3.5 μl, 100 ng/μl) were combined with 25 μl/well of the cell suspension in which overall sulfotransferase activity was to be determined in the wells of a microtiter plate. For negative controls, assay buffer was substituted for the cell suspension. The mixture was incubated for 20 minutes at 37°C, then 30 μl of malachite green was added and the mixture gently tapped. De-ionized water (100 ul) was added to each well, and color was developed. Optical density of each well was read and compared among the different wells. The amount of product formation was calculated using a phosphate standard curve, following subtraction of the reading of the negative control. The 3-inorganic phosphate released by the coupling phosphatase and detected by malachite green phosphate detection reagent, was proportional to the 3′-phosphoadenosine-5′-phosphate generated, thereby indicating the extent of the sulfotransferase reaction which utilized the sulfate group of PAPS.
Bhattacharyya S., Feferman L, & Tobacman J.K. (2014). Regulation of Chondroitin-4-Sulfotransferase (CHST11) Expression by Opposing Effects of Arylsulfatase B on BMP4 and Wnt9A. Biochimica et biophysica acta, 1849(3), 342-352.
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5

Profiling Sulfotransferase Enzyme Activity

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Recombinant sulfotransferases (sult1b1, 1c2, 1e1, 2a1, and 1a1) and the Universal Sulfotransferase Activity Kit (R&D Systems, Minneapolis, MN) were used according to manufacturer’s recommendations. Following analysis by plate reader, samples were analyzed by LCMS as described above to confirm that the sulfated product was indeed 4EPS. Cytosolic fractions from 50–200ug tissue containing endogenous sulfotransferases were extracted and tested for SULT activity on 4EP as previously described49 ,50 and analyzed by LCMS as described above.
Needham B.D., Funabashi M., Adame M.D., Wang Z., Boktor J.C., Haney J., Wu W.L., Rabut C., Ladinsky M.S., Hwang S.J., Guo Y., Zhu Q., Griffiths J.A., Knight R., Bjorkman P.J., Shapiro M.G., Geschwind D.H., Holschneider D.P., Fischbach M.A, & Mazmanian S.K. (2022). A gut-derived metabolite alters brain activity and anxiety behaviour in mice. Nature, 602(7898), 647-653.
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6

Quantifying Sulfotransferase Activity in NSCLC

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Sulfotransferase activity was detected with the Universal sulfotransferase activity kit (R&D # EA003) and measured according to the manufacturer's protocol. NSCLC cells were washed twice with normal saline and one time with 1X assay buffer. Then, the cells were lysed in 1% NP-40 in 1X assay buffer and sonicated. Next, 10 µg of total protein lysate was used to measure the sulfotransferase activity.
Chang W.M., Li L.J., Chiu I.A., Lai T.C., Chang Y.C., Tsai H.F., Yang C.J., Huang M.S., Su C.Y., Lai T.L., Jan Y.H, & Hsiao M. (2022). The aberrant cancer metabolic gene carbohydrate sulfotransferase 11 promotes non-small cell lung cancer cell metastasis via dysregulation of ceruloplasmin and intracellular iron balance. Translational Oncology, 25, 101508.
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7

Quantification of Sulfated Glycosaminoglycans

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Measurement of total sulfated glycosaminoglycans (GAGs) was performed, as previously described [14] . Briefly, the Blyscan TM assay kit (Biocolor Ltd, Newtownabbey, N. Ireland) was used for detection of the sulfated GAGs, based on the reaction of 1,9-dimethylmethylene blue with the sulfated oligosaccharides in the GAG chains.
Sulfotransferase activity was determined using the Universal Sulfotransferase Activity kit (R&D, Minneapolis, MN, USA), which assesses the sulfotransferase activity of all the sulfotransferases in the cells or tissue tested [14] .
Tzankov A., Bhattacharyya S., Kotlo K, & Tobacman J.K. (2022). Increase in Chondroitin Sulfate and Decline in Arylsulfatase B May Contribute to Pathophysiology of COVID-19 Respiratory Failure. Pathobiology : journal of immunopathology, molecular and cellular biology, 89(2).
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