TY - JOUR
T1 - A microtiter-based fluorescence assay for (1,3)-β-glucan synthases
AU - Shedletzky, Esther
AU - Unger, Christoph
AU - Delmer, Deborah P.
PY - 1997/6/15
Y1 - 1997/6/15
N2 - A high-throughput assay for UDP-Glc:(1,3)-β-glucan synthase(EC 2.4.1.34, UDP-glucose:1,3-β-D-glucan, 3-β-glucosyltransferase) from fungi and higher plants is described. The assay is performed in microtiter plates and is extremely inexpensive compared to other standard assays for these enzymes. The reduction in price is achieved by replacing the conventional substrate UDP-[14C]Glc with its nonradioactive counterpart, and the nonradioactive glucan produced is quantified as a fluorescent complex following specific interaction with the fluorochrome present in commercial aniline blue. In addition to a >100-fold reduction in cost, the assay is highly reproducible and nearly as sensitive as radioactive assays and has the additional advantages of increased safety and avoidance of the need for filtration and washing steps to collect the glucan product. As such, the assay is highly suitable for high-throughput screening for inhibitors of these enzymes.
AB - A high-throughput assay for UDP-Glc:(1,3)-β-glucan synthase(EC 2.4.1.34, UDP-glucose:1,3-β-D-glucan, 3-β-glucosyltransferase) from fungi and higher plants is described. The assay is performed in microtiter plates and is extremely inexpensive compared to other standard assays for these enzymes. The reduction in price is achieved by replacing the conventional substrate UDP-[14C]Glc with its nonradioactive counterpart, and the nonradioactive glucan produced is quantified as a fluorescent complex following specific interaction with the fluorochrome present in commercial aniline blue. In addition to a >100-fold reduction in cost, the assay is highly reproducible and nearly as sensitive as radioactive assays and has the additional advantages of increased safety and avoidance of the need for filtration and washing steps to collect the glucan product. As such, the assay is highly suitable for high-throughput screening for inhibitors of these enzymes.
UR - http://www.scopus.com/inward/record.url?scp=0031570761&partnerID=8YFLogxK
U2 - 10.1006/abio.1997.2162
DO - 10.1006/abio.1997.2162
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C2 - 9193713
AN - SCOPUS:0031570761
SN - 0003-2697
VL - 249
SP - 88
EP - 93
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -