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Glucan Synthetase Assay
adapted from Szaniszlo, P. J., Kang, M. S., and Cabib, E.
1984. Stimulation of ß(1>3) Glucan Synthetase of Various Fungi by Nucleoside
Triphosphates: Generalized Regulatory Mechanism for Cell Wall Biosynthesis. J.
Bacteriol. 161: 1188-1194. |
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Materials
Protocol
Results
Tips |
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Principle
and General Applications |
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Materials |
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5 mM UDP-[14C]glucose
75 mM Tris-HCl
0.75% (wt/vol) BSA
8% (vol/vol) glycerol
25 mM KF
10% trichloroacetic acid (TCA) |
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Protocol |
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- Mix the standard assay mixture (5 mM UDP-[14C]glucose
(2 x 105 cpm/µmol), 75 mM Tris chloride (pH 8), 0.75% (wt/vol) BSA, 8% (vol/vol) glycerol
(usually contained in the enzyme preparation), 25 mM KF, and enzyme (usually about 200 µg
of protein) in a total volume of 40 µl.
- Incubate for 40 min at 30°C.
- Precipitate glucan by adding 10% trichloroacetic acid (TCA).
Filter precipitate mixture through 25 mm glass fiber filters (type A/E) (Gelman
Science, Ann Arbor, MI).
- The incorporated radioactivity in the glucan can be measured
using a liquid scintillation counter.
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Results |
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Tips |
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This page updated on:
Tuesday, March 04, 2003 12:18:49 AM |
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